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METHIONINE METABOLISM IN FILARIAL WORMS
BY
Robert Asare Acquaah

Department of Biochemistry
University of Ghana, Legon
Date: September, 1980
Dr. K.K. Oduro, Internal Supervisor
&r. J.J. Jaffe, External Supervisor
m f v A
Or. P.H. Gyang, Member of Supervisory Committee
Thesis submitted to the Department of

Biochemistry, University of Ghana,
Legon in fulfillment of
the requirements for the
degree of Doctor
of Philosophy.
The experimental work for this thesis was
conducted at the University of Vermont, U.S.A.,
under the direct supervision of Or. J.J. Jaffe
of the Department of Pharmacology.

TABLE OF CONTENTS
TABLE OF CONTENTS 1
LIST OF TABLES iv
LIST OF FIGURES v
LIST OF PLATE vi
LIST OF ABBREVIATIONS vii
ACKNOWLEDGEMENTS 1
ABSTRACT 2
Chapter
LITERATURE REVIEW AND INTRODUCTION 3
A. Life Cycle of Filariae 3
B. Pathogenesis of Filariasis 4
C. Socio-Economic Aspects of Filariasis 5
D. Treatment of Filariasis 6
(a) Diethylcarbamazine 7
(b) Suramin 11
(c) Antimony Compounds 14
(d) Arsenic Compounds 15
(e) Metrifonate 15
(f) Mebendazole 16
(g) Levamisole 16
(h) Vector Control 17
(i) Sanitation, Urban Environmental

Improvement & Education 17
(ii) Chemical Control 18
(iii) Biological Control 19
(iv) Genetic Control 19

E. Metabolism of Filarial Worms 19
i
2. MATERIALS AND METHODS **
MATERIALS
METHODS 28
A. Delineation of Methionine Metabolic Pa thway in Filariae 28
B. Preparation of Extract 29
C. Enzyme Assays 30
(a) N ^- M e t h y l T H F :homocysteine S-Methy lt ra ns fe ra se 30
(b) Betaine:homocysteine S- me th yl tr an sfe ra se 30
(c) ATP:methionine S-Adenosylt ra ns fe ras e 30
(d) S-Adenosylhomocysteine ( A do M et ):homocysteine

S - me th yl tran sf erase 31
(e) S-Adenosylhomocysteine (AdoMet)hydrolase 31
(f) Cystathione-3-synthase 32
(g) Cystathione-^-lyase 32
D. Metabolic Fate of AdoMe t in Filariae 33
(a) Extraction of Protein 33
(b) Extraction of Nucleic Acids 34
(c) Extraction of Lipids 34
3. RESULTS 36
A. Biosynthesis of Methionine by Filariae 36
B. A bility of Filarial Worms to Metabolize Met hi on in e 37
C. Partial Characterisation of S-Adenosylmethionine:

Homocysteine S-Methyltransferase 38
(a) Int ra ce l1ular Localization of AdoMet:

Homocysteine S-Methy lt ra ns fe ras e 39
(b) Dependence of Enzyme A ctivity on Time and

Enzyme Concentration 39
(c) Effect of pH and Ionic Strength on Enzyme

Acti vi ty 42
ii
(d) Effect of Temperature on Enzyme Activity. 42
(e) Requirement of Assay. 42
(f) Effect of Divalent Metal Ions and EDTA. 46
(g) Kinetic Studies. 47
(V) Effect of Inhibitors 47
D. Metabolic Fate of AdoMet in Filariae 50
4. DISCUSSION AND CONCLUSION 58
A. Folate Metabolism 58
8. Methionine Metabolism 59
(a) N®- MethylTHF:Homocysteine
S-Methyltransferase and Betaine:

59
Homocysteine S-Methy1 transferase.
(b) ATP .-Methionine S-Adenosyl transferase. 63
(c) Metabolic Functions of S-Adenosylmethionine. 65
(d) AdoMet:Homocysteine S-Methyltransferase. 73
C. AdoMet-Mediated Methyl transferases as
Potential Targets in the Chemotherapy of
Parasites. 81
REFERENCES 88
ii i
TABLE
1. Biosynthesis of Methionine by Filariae ^
I. Ability of Filarial Worn^ to Metabolize Methionine
3. Specific Activities of Some Filarial Methionine
Metabolizing Cnzyrces.
4. Partial Purification of Filarial Adcêt iHcwoc'/steine
S-Methyltransferase,
5. Intracellular Localization of AdoMetsHomocystelne
S-Methyltransferase.
6. Requirement of Assay <v Filarial Adoâ*ilJonPcysteine
S-Methyltransferase
7. Effect of Metal Ions on Filarial AdoMet:Homocysteine
8. Effect of EDTA on Fllflnal AdoMet;Homocysteine
9. Kinetic Parameters cf Filarial AdoMet:Homocysteine
S-Methyltrans ferase.
10. Distribution of Radioactivity after Incubating Adult
D ,Imifiitis in the Presence of (CH^-^C)Methionine.
II, Some Examples of Transmethylation Reactions Involving AdoMet*

LIST OF FIGURES
The Chemical Structure of Diethylcarbamazine. 9
The Chemical Structure of Suramin. 13
The Chemical Structures of Levamisole,

4-Melami nyl-l-( methyl olcyclo (ethyl enedithiastibina) 15
benzene, and Pentylthiarsaphenylmelamine.
The Chemical Structures of Metrifonate and Mebendazole* 16
Time-Course of Formation of Methionine by

AdoMet:Homocystei ne S-Methyltransferase. 40
Formation of Methionine by AdoMet:Homocysteine

5-Methyltransferase as Function of Enzyme Amount. 41
Effect of pH on AdoMet‘
. Homocysteine S-Methyltransferase. 43
Effect of Ionic Strength on AdoMet:Homocysteine

S-Methyltransferase. 44
Effect of Temperature on AdoMet:Homocysteine

S-Methyltransferase. 45
Lineweaver-Burk Plot of AdoMet Keeping Homocysteine

Concentration Constant. 48
Lineweaver-Burk Plot of Homocysteine Keeping

AdoMet Concentration Constant. 49
Radioactivity Distribution in Thin Layer Chromatogram

of Phospholipids. 52
Autoradiography of Filarial Phospholipids. 53
Radioactivity Distribution in Thin Layer Chromatogram

of Protein Hydrolysate. 56
Major Pathways for Methionine Metabolism in Mammals. 57
v
LIST OF PLATE
PLATE Pafle
1 Photograph of Iodine-Developed TLC of Filarial
Phospholipids, 55
vi
L I S T OF A B B R E V I A T I O N S
AdoHcy, AdoMet S-Adenosylhomocysteine, and - methionine
ADP, ATP Adenosine 5'-di-, and triphosphate
BHC Ganrna-benzenehexachloride
812 Cyanocobalamin
BSS Balanced salts
C Carbon
°C Degree Centrigrade
Ca Calcium
Cd Cadmium
Co Cobalt
CoA Coenzyme A
co2 Carbon dioxide
CH? Methylene
CH3 Methyl
Cone. Concentration
cpm Counts per minute
0 Dextrorotatory
DL Racemic mixture
DDT Dichiorodi phenyltri chioroethane
DEC Diethyl carbamazi ne
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
d.p.m. Disintegration per minute
DTT Dithiothreitol
EC Enzyme Commission
EDTA Ethylenediaminetatraacetic acid
vi i
FAD Flavin adenine dinucleotide
FMN Flavin mononucleotide
g Earth's gravitational field, 980.6 cm/sec^
GSH Reduced glutathione
h Hour
HC1 Hydrochloric acid
KC1 Potassium chloride
KCN Potassium cyanide
Km Michael is constant
L Levorotatcry
M Molar
mC1 Milli curie
MCP Methyl-accepting Chemotaxis Protein
Mel W Pentylthiarsaphenylmelamine
MEME Minimum essential medium, Eagle
Met Methionine
mg Milligram
MgCI 2 Magnesium chloride
mtn Minute
ml Mill n i t r e
mmol Millimole
Mn Manganese
mRNA Messenger RNA
MSbB 4-Melaminyl-l- (methylolcylo (ethylenedithiastibina)
benzene
WA 5'-Methylthioadenosine
N Nitrogen
viii
NaCl Sodium chloride
NAD+ ,NADH Nicotinamide adenine dinucleotide and its reduced form
NADPH Reduced nicotinamide adenine dinucleotide phosphate
NaOH Sodium hydroxide
nCi Nano curie
NH 4OH Anmonium chloride
Ni Nickel
PDE.PME Phosphatidyl-moitoethyl-, and -dimethyl-ethanolamlne
pH - Log H+ concentration
P(i) (Inorganic)phosphate
PPO 2,5-Diphenyloxazole
Rb Rubidium
RNA Ribonucleic acid
RNase Ribonuclease
S Sulphur
sec Second
S042 ” Sulphate
SSC NaCl and sodium citrate
TCA Trichloroacetic acid
TCA cycle Tricarboxylic acid cycle
THF Tetrahydrofolate
TKM Tris, KC1 and MgCl 2
"N-C Thin layer chromatography
Tris( hydro xymethyl )aminomethane
^RNA Transfer RNA
^ Antimony a, a-dimercaptosuccinate
^ Uridine triphosphate
ix
uv Ultraviolet
V Volume
Vmax Maximum velocity
w Weight
Zn Zinc
ul Microlitre
um Micrometre
% Percent
X
ACKNOWLEDGEMENTS
My thanks are primarily due to my external supervisor Dr. J.J. Jaffe
of the Department of Pharmacology, University of Vermont, for his
helpful advice and encouragement throughout this research. I should also
like to thank him and his wife for making my stay in Burlington enjoyable.
The greatest indebtedness that I owe is to the World Health
Organization for the financial supporc without which this project could
not have been undertaken.
My indebtedness also goes to Prof. D.A. Bekoe, the Vice-Chancellor,
University of Ghana, Legon and Prof. G.S. Asante, the Head of Department
of Biochemistry, Legon for going through all the administrative tedium
of setting me on a career course. I am also grateful to my supervisory
conmittee members in the Department of Biochemistry, University of Ghana,
Legon: Drs. K.K. Oduro (Internal Supervisor), F.N. Gyang and S. Asante-Poku
for their valuable suggestions.
I should further like to express my appreciation and thanks to
Dr. J.C.W. Comley, Mrs. L.R. Chrin and Mrs. R. Smith for their immense
contribution to this work. Also deserving gratitude are Drs. Alan Eastman,
Lazarus Durkwa, Charles des Bordes, and Tshimanga N'Zagi, Mr. Donald Taylor,
the entire faculty members of the Department of Pharmacology especially
Drs, J,0. McCormack and R. Newman, the staff of the International Student
Office, University of Vermont, and Mr. Gabriel B. Fosu of Brown
University, Providence, Rhode Island. Finally, but not the least, much
thanks go to Miss Gail Fairbanks and Mr. Kofi Gyau for typing the manuscript.
}\\‘
STRACT
Adult filariae apparently lack the vitamin B I2 -d ependent and
-independent methyl transferases for the de novo synthesis of m et hionine
and seem to meet their requirement of this amino acid from an exogenous
source and the activity of S - a d e n os y lm et hi o ni ne ( Ad o Me t) :homocysteine
S-methyltransferase. The properties of filarial A do M et :h om oc ys te in e
methyl transferase were similar to the analogous microbial enzyme.
However, adult filariae possess the enzymatic c ap ability to m et abolize
methionine to cyst(e)ine.
When incubated in the presence of L - f C H ^ - ^ C j m e t h i o n i n e to
induce them to synthesise AdoMet, adult Dirofilaria immitis incorporated
the radiolabel into phospholipid and protein fractions.
The significance of filarial methionine me tabolism especially
with regard to its suitability as a potential target for antifilarial
chemotherapy is discussed.
Capter 1
LITERATURE REVIEW AND INTRODUCTION
A. Life Cycle of Filariae
Filariae are parasitic nematodes of the Order Filariidea which
cause filariasis in millions of people in tropical countries (1).
Although filariasis usually refers to infections with Wuchereria
b an cr of ti , Bruqia malayi and B r u g ia t i m o r i , in the present context it is
used more broadly to include those of Onchocerca v o l v u l u s , Loa l o a ,
Mansonella o zz a r d i , Dipetalonema p e r s tans and Dipetalonema s t r e p t o c e r c a .
Filarial worms pass through five developmental stages in their life
cycle. The adult or fifth-stage worms take months to mature, are long-
lived and do not undergo asexual multiplication in the definitive host.
After mating the viviparous females discharge, according to the species,
numerous sheathed or unsheathed microfilariae or first-stage larvae.
Except for the microfilariae of 0 . v olvulus and D.streptocerca which
invade the skin, the others circulate in the peripheral blood, and they
may exhibit different kinds of circadian periodicity (2). When ingested
by the appropriate insect vector with its blood meal, the m ic ro filariae
of all species migrate either to the flight muscles, the Malpighian
tubules, or the abdominal fat bodies, where they m et am or pho se intra
cellularly, without multiplying, into the infective filariform (tnird-
stage) larvae. Vectors of W.bancrofti belong to the genera C u l e x , Aedes
and Anopheles while Mansonia mosquitoes are the main secondary hosts of
Bnjgj_a. Various species of Simul ium , Chrysops and Cu 1 icoides are the
respective intermediate hosts of O n c h o c e r c a , Loa and Pi p e t a l o n e m a . The

Infective larvae migrate to the salivary gland in the head of
the insect vector, and enter the definitive host through the proboscis
when the Insect takes its blood meal. Once in the final host the larvae
migrate to their preferred sites: skin (Onchocerca, L o a , D.streptocerca),
lymphatics (Wuchereria, Brugia), or body cavity (Hansonella, D.perstans)
and there undergo two moults before they become mature parasites.
B. Pathogenesis of Filariasis
Characteristic features of onchocerciasis include nodulization,
onchodermatitls, hanging groin, hernia, elephantiasis and eye lesions (3).
The severity of the disease is a result more of reaction to the presence
of microfilariae than to the adults, The inception of onchocerciasis
might go unnoticed because the adults which live 1n the subcutaneous
tissues may be unpalpable (4). As the disease progresses distinctive
nodules or "onchocercomas" appear in which are found dead or dying
worms (5). Onchodermatitis, which 1n some pattents may be asymptomatic,
is prominent at this stage. Characteristically, there are pruritis,
papulosis, dermal thickening and eventually pachydermia which gives a
senescent and emanciated look to the patient, Chronic skin lesions
cause depigmentation called leopard skin especially over the legs.
Pachydermia predisposes usually adult males to hernia, pseudo-
adenolymphocele or "hanging groin". The latter 1s a pendulous sac
containing inguinal or femoral lymph gland which is known to cause
scrotal elephantiasis 1n some endemic areas of Africa.
Ocular onchocerciasis is the most devastating aspect of this
form of fllarlasis; it is an inflamnatory reaction in the eye
tissues to dead microfilariae. Conjunctivitis, corneal sclerosing keratitis

and anterior uveitis are comnon features. Invasion of the posterior
chamber of the eye may lead to the atrophy of the optic nerve and the
retina (3,6,7), resulting in “river blindness".
Although infection with Wuchereria and Brugta may be asymptomatic
in some individuals, the acute phase is marked by lymphangitis, fever,
lymphadenitis, lymphoedema and elephantiasis of limbs, genitalia and
breast. Cryptic infection with microfilariae 1n especially the lungs
and hyperplastic lymph nodes is a common aetiology of tropical
eosinophilia (8-13).
"Calabar swelling", subcutaneous and transocular migration of adult
worms and hypereosinophilia are the main symptoms of loaiasis. Occasionally
there might be albuminuria which may be aggravated by dlethylcarbamazine
treatment. Invasion by microfilariae of the cerebrospinal fluid may
predispose persons with high microfilaremia to meninoo-encephal itis(14).
Recent evidence (7) has associated the once considered non-pathogenic
dipetalonemiasis and mansonelliasis with eosinophilia, papulosis and
pruritis.
C. Socio-Economic Aspects of Filariasis
The pathogenesis of the different forms of filariasis reveals that
the disease is capable of producing severe debility, the socio-economic
consequences of which must be enormous.
The economy of countries in the filariasis-endemic regions of
the world is primarily agricultural, relying heavily upon manual
labour, malnutrition and infectious diseases, among other factors,
reduce the quality of the labour force resulting in a lower output.

In many parts of West and equatorial Africa, more than 50* of the
inhabitants are infected with onchocerciasis; 30% have amblyopia and 4
10% are blind (15). In some villages of Upper Volta and Northern Ghana the
prevalence of blindness in adult males may reach 30-40%, whereas the
rate of blindness in onchocerciasis-free zones is between 0.5-1.0%. The
average proportion of blind persons in endemic areas is 10%, as a gainst
0.2% in regions free of onchocerciasis (16).
Morbidity due to onchocerciasis has led to depopulation of fertile
land near rivers where Simulium damnosum breeds. It is estimated that

9 .
65,000 km*" of the Volta River basin with a potential annual agricultural
output of about US $30 million are deserted (17). To urism which provides
some developing countries with substantial foreign exhange is also
seriously affected because the tourist attractiveness of endemic areas
is considerably reduced.
Filariasis, like other tropical diseases, is therefore partly
responsible for impeding progress and economic development in many
developing countries. Its eradication or control would have a trem en
dous socio-economic impact.
D. Treatment of Filariasis
Chemotherapy and chemoprophylaxis play an important role in the
control of some parasitic infections. Although a vaccine for malaria
perhaps is in sight (18), for the majority of the parasitic diseases a
practical method of immunization against them may only be realized in
the distant future. Until then, drugs will continue to play a significant
role in the control and treatment of these infections.

Di ethy1carbama zi ne (1-diethyl - arbamyl-4-methyl piperazine) and
suramin (trisodium salt of 8 , 8 '- ( J ' ', 3 * ' ‘

- u r e y l e n e b i s ^ 1 1’‘
-benzamido-
4 11 1 1-m et hy lbenzamido))-bis-1,3,5 ,- na ph th alenetri s u lphonic acid) are the
only effective drugs presently available for the treatment of human
filariasis, and knowledge about them, including their clinical phar ma
cology, were recently thoroughly reviewed by Hawking (19,20).
(a) Diethylca rba ma zi ne : The antifilarial activity of d ie t hy lc arb am az in e
(DEC) was discovered by Hewitt and colleagues (21) in 1947 using a
cotton rat, Siqmodon h i s p i d u s , naturally infected with L i tomosoides
carini i. It was found that neither DEC nor sera derived from treated
animals exerted any antifilarial action in v i t r o . By contrast a profound
and rapid effect was obtained when DEC was administered intravenously to
L .carinii-i nfected cotton rats; there was an 80% reduction in m i c r o
filaremia within two minutes (22,23). DEC is similarly potent in vivo
against the microfilariae of W . b a n c r o f t i , B . m a l a y i , 0 . vo lv u lu s ,
D. streptocerca, L.loa and D i rofilaria imrnitis ( 2 , 3 , 6 , 9, 1 4, 2 3- 2 5) .
It was established by a number of investigators that DEC had n?
activity against adult filariae in v i t r o . The susceptibility of adult
worms to DEC in vivo was found to be more variable than that of the
microfilariae and appeared to be species-dependent. Hewitt et al. (21)
reported that DEC was a macroftlaricidal aqent aqainst L.c a r i n i i , but this
finding could not be confirmed by Hawking et aj_. (22). The latter
investigators suggested the duration of infection as the cause of the
anomaly; since Hewitt's group used rats with comparatively old infections
and death of the worms might have been due to senility or to a host
immune response. Treatment of patients infected with 0.volvulus using
ordinary dose regimes of DEC showed no adverse consequences on the adult
worms (26); however, some of the adult filariae were destroyed but only
after prolonged intensive treatment (21,27). It was reported that adult
L.Loa, D.perstans and W.bancrofti could be killed by DEC as evidenced by
the prolonged absence of detectable macrofilaremia after administration
of the drug (23,28-30). Adult B.malayi and D.streptocerca are also
susceptible to DEC, although a high disage is required to kill the
latter (8,10,14, 31-33).
Absorption & Distribution; Although DEC is normally given orally, it
can be applied topically over nodules in onchocercal cases (20). Whatever
the route of administration the drug is rapidly absorbed. According to
estimation of Rees et al.(34) after a single oral dose of 200-400mg to
man, a peak plasma level was reached within two hours; after a dose of 200 mg
the plasma half life was 8.1 + 3.5 hr and 11.7 + 2.3 hr after 800 mg of
DEC. Although a maintenance level could be attained with large daily
doses, there was no tendency for the drug to accumulate. DEC 1s apparently
widely distributed, with liver, kidneys, adrenal glands, muscle,
gastrointestinal tract, salivary glands and brain exhibiting high levels (20).
14 , .
Metabolism & Excretion: Using C-labelled DEC, Bangham (35) showed
that the piperazine ring was entirely eliminated within 24-30 hr in the
urine. Analysis of urinary metabolites by paper chromatography and ion-
exchange column chromatography following intravenous administration of
DEC showed that 10-20% of the compound was excreted intact, 8-15% as
diethylcarbamylpiperazine, 2-5* as methylpiperazine and 1.62 as piperazine

Figure 1
The Chemical Structure of Diethylcarbamazine
I
Ol
O
and the remaining 60% as unidentified compounds. Subsequent investi
gations by Faulker and Smith (20) revealed the identity of the unknown
metabolites as diethylcarbamazine-N-oxide, 50%, and 1- e th yl ca rb am yl -4-
niethyl piperazine, 23%. Although me ta bo li sm is rapid and extensive, no
metabolite so far isolated appears to have a sufficiently profound
effect to account for the extraordinary speed of action on the circulating
microfilariae.
flode of A c t i o n : The exact mode of action of DEC is uncertain. Current
evidence suggests that it may be exerting an opsonizing effect either by
causing exsheathment or by damaging the cuticle and thereby unmasking
antigenic determinants of inicrofi1ariae and susceptible adult worms leading
to phagocytosis by the host's immune system (23,27,35). Histological
studies on cotton rats infected with L .carinii have revealed that the
action of DEC in vivo is associated with aggregation of the micro fi lar ia e
in the liver where they are trapped by phagocytes leading to their
eventual dissolution (23,37,38). Implicit in this opsonin-like action
might be an immunological response since it has been observed that
microfilariae in serous cavities were apparently not destroyed by
DEC (24). Current work in experimental filariasis suggests that immunity
may be a prerequisite for the expression of the microfilaricidal activity
of DEC. Kobayashi et aj[. (37) found that the drug did not affect
microfilariae of L.carinii injected into normal cotton rats or circulating
larvae emerging from adult worms transplanted into naive hosts. In the
latter case, however, microfilariae were cleared from the circulating
blood fifteen days after t r a n s D l antation of m a t u r e worms. Treatment of
cotton rats with immuno-suppressive agents followed by DEC, however,

10
gave inconclusive results. DEC administered simultaneously with a n t i
lymphocyte serum to infected cotton rats was also ineffective. DEC
treatment was efficacious only after termination of a n t i lymphocyte serum
treatment.
because DEC is a piperazine derivative, it has been postulated that
this drug may possess antihistaminic properties as was demonstrated in a
series of related compounds (39-42). On the other hand, experiments
with rats and calves suggest that DEC rather than being antihistaminic
may be inducing the release of vasoactive mediators, including histamine (43).
An action of DEC on the neuromuscular system is also possible
particularly as acetylcholine and cholinesterase activity have been
detected in filariae (44). Piperazines have been shown to hyperpola;'ize
isolated muscle cells of Ascaris lumbricoides var. suum with co nsequent
flaccid paralysis, the effect of which can be reversed by adding acetylcholine
(45-49).
Toxici t y : DEC is not without si de -e ff e ct s. Two types of toxic reactions
are noted in DEC therapy, those related to the direct effects of the
drug and those secondary to the death of the m i c r o n 1ariae and/or macro-
filariae.
In uninfected persons, DEC can cause gastrointestinal disturbances
especially if administered before meals. Other documented adverse
reactions include headache and drowsiness.
In patients infected with filariae the side-effects are variable
with the severity depending upon the form of filariasis and the w or m
burden. Side effects are particularly prominent in onchocerciasis (1 3 )

11
and include swelling, oedema, pruritis, hyperpyrexia and inflammatory
reactions, the latter complicating the treatment of ocular
onchocerciasis (50,51).
(b) Suramin: In 1920 Bayer Introduced suramin for the treatment of human
trypanosomiases. Its filaricidal efficacy particularly against 0.volvulus
was discovered in 1945 by Van Hoof and colleagues in Zaire (then Congo)
during field trials (23).
Contrary to the findings of Hawking (23), those of Lammler et a l .(52)
indicated that suramin could kill L.carinii in Mastomys natalensis
within six weeks. The two weeks observation period other workers
had utilised was too short a duration for any effect to be discernible.
Infective and other immature stages of L.carinii are very sensitive
to suramin (19). Subcutaneous injection of 40 mg/kg/day suramin for
five days starting one, two, or four weeks after infection completely
removed all traces of infection. Lower doses given for five days
inmediately after infection did not kill all the worms.
There was also a delayed response by adult 0.volvulus and W.bancrofti
to suramin. Female worms were more susceptible than the males, probably
due to their more active metabolism (53). The action of suramin on the
other filarial parasites of man is unknown.
Direct microfilaricidal activity of suramin is unusual, although
microfilariae of 0.volvulus are eventually cleared from the body possibly
by living out their normal life span (10-18 months), the macrofilaricidal
effect obviating replenishment. The development of infective larvae of
0.volvulus in chimpanzees is likewise terminated by suramin (19), In

12
two human volunteers, however, n<; chemoprophylaxis could be d e m o n s tr: ted
probably due to the short ob servation period (54).
With the possible exception of Simu 1 iuni d am no sum (the vector species
for 0 .v o l v u l u s ) no effect of suramin on the develop men t of mi cro fil ari ae
in the other arthropod hosts, mosqu it oes , tabanids and midges, has been
reported. In the case of Simulium the apparent microfilaricidal effect
is thought to have been exerted via the suramin- tre at ed patients on
whose blood the vectors fed (55).
Abs or pt io n & D i s t r i b u t i o n ; The limited intestinal a b s o r p t i o n of suramin
when given by mo ut h and the acute local i n f l a m m a t o r y react io n f oll o wi ng
s ub c u t a n e o u s or i nt r a m u s c u l a r i n j e ct i on has made the i n tr ave no us route
the practical co ur s e of a d m i n i s t r a t i o n .
Af te r i nt ra v en ou s in je c ti o n su r am in c o mb i n e s r a p i d l y witi'i s e ru m
pr ot e in s, and most of it c i r c u l a t e s in the blood in this bo un d fcrm.
Some of it, p r o b a b l y c o mb i ne d with p r ot ei n, is taken up by the cells of
the r e t i c u l o e n d o t h e l i a l system. Its p r e s e n c e in the e p i t h e l i u m of the
p roximal c o n v o l u t e d t u bu l e s of the k i d n e y has a lso been d e m o n s t r a t e d (19).
Metabolism & Excretion: A l t h o u g h e x c r e t e d by the k i dn e ys , m o s t of an
a d m i n i s t e r e d d os e p e r s i s t s in the b o d y for a p r o l o n g e d p eri od , d u e to
its e x t e n s i v e b i n d i n g to p l a s ma p r o t e i n s . A p p a r e n t l y s u r a m i n do es n ot
u n d e r g o any fo rm of m e t a b o l i c t r a n s f o r m a t i o n in the body, s u g g e s t i n g
that its c h e m o t h e r a p e u t i c a c t i v i t y is due to the i n t a c t m o l e c u l e .
M od e of A c t i o n : The s lo w o n s e t of s u r a m i n ' s m a c r o f i l a r i c i d a l activity,
w i t h a l a t e n c y of 5-6 wk, parallels its d e l a y e d trypanocidal action

Figure 2
The Chemical Structure of Suramin
S u r a m i n
13
where 1t is effective only after six cell divisions. In trypanosomes,
it is believed that suramin primarily inhibits DNA and RNA polymerases
probably by bridging the active sites (19). The presence of multinucleated
trypanosomes among those exposed to suramin also suggests that cytoplasmic
cleavage is Inhibited. The enzymatic mechanism responsible for this
subdivision 1s thought to be sensitive to suramin as has been demonstrated
in the bacterium Streptococcus foecalis (56).
Sensitivity of these important processes to suramin might explain
Its antifilarial action. Since it is only the reproductive cells which
are actively multiplying in the macrofllariae, it is likely that they
would be the sites of suramin's action. In addition, the inhibitory
action of suramin on some important key metabolic enzymes may further
serve to augment its primary effect. Suramin,was found to inhibit
hexokinase (19), glycerophosphate dehydrogenase and glycerophosphate
oxidase (252), Jaffe and colleagues (57) have found that dihydrofolate
reductases from four species of adult filarial worms, including 0.volvulus,
were sensitive to inhibition by suramin 1n the 2-10 uM range. Suramin
was also found to be a potent inhibitor of two other folate-related
enzymes from adult Brugia, 10-formyltetrahydrofolate dehydrogenase and
5-formyltetrahydrofolate cyclodehydrase (58).
Toxicity: This has been studied in depth in patients with trypanosomiasis.
The Immediate reaction to an intravenous injection of suramin
characterized by nausea, vomiting, sweating and/or coma, is followed by
a reaction with the following features: hyperpyrexia, photophobia,
transient conjunctivitis, gastrointestinal problems and nephrotoxicity (19,23).
Albuminuria, exfoliative dermatitis, prostration, chronic diarrhoea and

14
inflammatory reactions are some ol the severe side effects associated
with suramin therapy. The allergic reaction is triggered by the sensitization
of the host to the dead filarial worms* often predisposing some ocular
onchocerciasis patients to optic atrophy (51).
DEC is the drug of choice for mass chemotherapy. However, its side
effects are a stumbling block to mass treatment (14). New antifilarial
drugs with higher efficacy and fewer side effects are urgently needed.
Meanwhile, a wide variety of presently available parasitic druqs have
been tested or are being reevaluated as antifilarial agents.
(c) Antimony Compo und s: In addition to their use in the treatment of
trypanosomiasis and schistosomiasis, antimony-containing compounds were
shown to be effective against D.immi tis in dogs and W . bancrofti in man.
Neostibosan was found among various antimony derivatives to be potent
against L.carini i in cotton rats and human If.bancrofti. It was also
effective against L.loa but not against 0 . volvulus (59,60). Friedheiin (61)
also showed that MSbB (4-melaminyl-l-[methylolc ylo (e th yl en ed it hi as tib in a) ]-
benzene) was effective against human W . b a n c r o f t i . Extensive field
trials by Duke (62) on patients in West Africa with Q .vo lvulus revealed
the efficacy of TWSb (Antimony a ,a-dimercaptosuccinate) and M S b 6 in the
treatment of onchocerciasis. However, this promising lead could not be
exploited owing to the low margins of safety of these antimony-based
c om po un ds .
Figure 3
The Chemical S t r u c t u r e s of Levamisole,

i Melaminyl-l-(methylolcyclo(ethy1ened1th1ast1blna)
benzene, and P e n t y l t h i a r s a p h e n y l m e ! a i m ne.
L e v a m i sole
4 - M e la m in y l - K m e t h y lo lc y c l o (ethylenedithiastibina )
b e nz e n e
HoN KOOC
\C ■N.
H
I
/ \
I
M—
\ - n / \S — CH
Ho N
/
KOOC
Pentylthiarsaphenylmelamine
15
(d) Arsenic Compounds: A r s e n i c a l also are efficient filaricides. Mel U
(Pentylthiarsaphenylmelami ne) introduced by Friedheim (63,64) was found
to be macrofilaricidal against W.bancrofti and Q.volvulus in man, but
its sometimes fatal s ide - e f f e c t s , however, precluded its further
application. Thiacetarsamide killed macro- and microfilariae in patients
infected with IJ.bancrofti. It was lethal to microf i lariae of li. bancrofti
and 0 .volvulus in vi tro and is effective against adult D.immitis in
dogs. Like Mel W, its clinical application was precluded because of
its toxicity.
(e) Metri fona te: Dimethyl- (2, 2, 2-trich ior o-1 -hy d r o x y e t h y l )- p h o s p h o n a t e ,
originally developed as an insecticide, can kill blood fluke trema-
todes, particularly Schistosoma h ae ma t o b i u m , as well as other helminths.
It is a potent anticholinesterase in helminths as well as in insects.
Metrifonate is a powerful microfilaricide against L .carini i in Mastomys
natalensis (52,65). It is macrofilaricidal against Dipetalonema witeae
M.natalensi s (37), but against D .wi teae infection in the jird, fieri ones
pe rsicus, metrifonate has no therapeutic efficacy (37). Denham and
associates showed that the drug was macrofilaricidal against Crugi:
pahanqi in cats ( 66 ).
Its anti-onchocercal action was first reported by Salazar-Mallen (67)
who found that a dose of 10 mg/kg daily for 6 days reduced the microfi lare-mia
of 0 . volv u l u s . In experiments with chimpanzees infected with 0 . volv ulu s ,
Duke (68-70) confirmed its m i c r o f i 1aricidal activity and also showed
that a sustained high dose of 22 mg/kg daily for 6 days had no effect on
the adult worms, despite noticeable host toxicity.

Figure 4
The Chemical Structures of Metri fo nat e and Mebendazole
0 O H
II I
c h 3o ------ P -------- c - CCI 3
1 I
CH 3 0 H
M et rif o n a t e
N
\C NHCOOCH3
N
M e bendazole
16
(f) M e b en da z ol e : Mebendazole [5 (6 )- be nzoyl-2-benzimidazole carbamate]
is a broad-spectrum anthelmintic ihich interferes with the microtubular
apparatus (71). The supposition is that the disruption of the m ic ro
tubules leads to a breakdown of the transport and secretory mechanisms
within nematode intestinal cells which in turn leads to an impairment of
digestion and absorption of nutrients, thereby affecting the synthesis,
uptake, transport and accumulation of basic substances. This mode of
action can account for the earlier reported inhibition of glucose uptake
by mebendazole (72).
Mebendazole was shown to be active against microfilariae and
adults of L.carinii and D.witeae in jirds but was without effect on
B.pahangi in jirds or 0 . volvulus in chimpanzees (73,74). Maertens and
Wery (75) also found the drug ineffective against 0. vol vulus in hurars
when given as an oral dose of 100 mg twice daily for 6 days. Micro
filariae of D.perstans in humans were susceptible to m ebendazole -after a
two-week treatment.
(g) L ev a mi s ol e: Levamisole is the levo-rotating isomer of 2,3,5,6-tetra-
6-phenylimidazo(2,l-b)thiazole. It is a broad-spsctruni anthelmintic
agent which most probably acts by stimulating ganglionic structures in
nematodes, resulting in a depolarizing type of neur om us cu lar b l o c k a g e
(76,77). It is also a powerful inhibitor of fumarate reductase in
vitro (71). In addition levamisole is an iminuno-stimulant (73-80),
although its antiparasitic properties apparently do not
depend on this action (81).
Lammler and associates (65) found levamisole to be fi laricida't
against L.ca rin ii in M.natale n s i s . It was also found to be effective

17
against B.malayi> B.pahangi, W.bancrofti and D.witeae (82,83). The
therapeutic Index of levamisole in the treatment of experimental oncho
cerciasis was relatively low; though filaricldal, a dose of lOmg/kg/day
given intramuscularly for 15 days to a chimpanzee, was intolerable (74).
In human trials, a single dose of 120 mg/week for three weeks had no
antifilarial activity in ten patients infected with 0.volvulus. Reduction
in microfilaeremia was observed in patients with B.malayi during a
course of levamisole treatment, although upon cessation of chemotherapy
there was an upsurge in the density of microfilariae.
(h) Vector Control: Where treatment with drugs is not feasible, the
control of filariasis must depend on vector control, using such methods as:
(1) Sanitation. Urban Environmental Improvement & Education: The
ideal strategy in vector control is the modification of the habitat,
thereby preventing breeding of the vectors. In particular, poor environ
mental sanitation results in the creation of ecological conditions
favourable for rapid multiplication of the vector population (84,85).
Increase in density of Culex pipiens fatigans in many urban centres of
the tropical world 1s a reflection of sanitary deterioration. Where
elaborate sanitation systems have been introduced, the mosquito population
has declined with a considerable reduction in the incidence of filariasis (14)•
Health education progranmes aimed at raising the awareness of the
inhabitants in these regions to the values of sanitation would be important.
However, health education, while effective, is a slow process and requires
such elaborate training of the affected people that it is often
viewed with Indifference. For the time being, therefore, reliance
must still be placed for the most, part on other effective approaches.
18
(1i) Chemical Control: The use of Insecticides in parasite vector
control has been extensively investigated, especially in connection.with
malarial eradication programmes. Because of its long-lasting effects,
dichlorodiphenyltrichloroethane (DDT) has been widely used to kill
adult insects. However, DDT is now ineffective in C.p.fatigans
control (86), as repeated application has caused the emergence of
resistant strains. Development of resistance to other chlorinated
hydrocarbon insecticides such as # -benzenehexachloride (BHC) and
dieldrin has also been reported (86). Organophosphorus compounds (e.g.,
fenthion, chlorpyrifos and chlorfenvinphos) were found to be effective
against these DDT-resistant strains, although resistance to the
organophosphate compound malathion in C.p. fatigans from Douala, Cameroun,
has now been reported (86).
In some instances, killing the larval insect stages may be preferential
to adulticiding. For example, although the latter approach has usually
been adopted for endophilic vectors, exophilic biters are better
controlled with larvicides. Systematic application of 0.1 ppm DDT to
streams in selected endemic areas of onchocerciasis in East Africa was
potent in controlling Simulium (37,86). High-spreading oils e.g. Malariol HS
(Shell Oil Co) has also been used. Mansonia larvae have been controlled
by spraying Pistia stratiotes, the water lettuce, with the herbicide
Slmazine, since Mansonia larvae are in close association with this aquatic
plant from which they derive oxygen. Like adulticides, resistance by
larvae to some of these larvicides is known (86).

19
(iii) Biological Control: Becau e of the actual or potential harm to
the environment by chemical agento used for vector control in addition
to the development of resistance to some of these in s e c t i c i d e s , the use of a l t e r
native methods .has become imperative.
Effective agents for the biological control of filarial vectors
include certain species of larvivorous fish such as Gambusia a f f i m ' s ,
Lebistes reticulatus and Nothobranchi us taenipyqus (87,88). Other
promising agents are: a strain of Bacillus thurinqiensis ; certain
fungi, such as Coslomoinyces s t e q o m y i a e , Laqenidium g i g a n l e u ^ , Metarrhizii;=
anisopliae and the mermithid nematode, Reesimennis n i e l s e n i .
The wide-spread application of biological agents for vector control,
though, poses potential direct and indirect hazards to man (83).
(iv) Genetic Co nt r o l : Research efforts directed towards the develop
ment of sterile-male techniques and other forms of genetic rani pulation
for the control of mosquito and other vectors of disease seem encouraging.
Some experimental results are encouraging for including such methods in
future control programmes (37,89,90).
E. Metabolism of Filarial W o r m s : Currently employed chemotherapeutic
agents in filariasis leave much to be desired. Effective and less toxic
filaricides are urgently needed. Like other helminth pa r a s i t e s sadult
filariae do not undergo asexual multiplication within their mammalian
hosts, but instead mate to produce offspring which undergo further
development within intermediate hosts. Therefore it might be expected
that if a new efficacious macrofilaricide were developed, not only would
it be a much needed curative agent but as a consequence of its wide
application the transmission cycle of filariae would be interrupted.

20
Such a drug could be found by empirical screening of all sorts of
chemical compounds. Alternatively, basic knowledge of the intermediary
metabolism of filarial parasites might reveal biochemical pathways which
could be blocked selectively by appropriate drugs.
Except for the pathways of carbohydrate metabolism and energy
production, the metabolism of filariae generally has not been a research
priority. Carbohydrates apparently form the major energy reserve of
parasitic helminths, including filariae. It is not surprising that
compounds whose mechanism of action lies in the interference with
carbohydrate metabolism, such as antimonials and arsenical, have played
a significant role in the control of a number of helminthiases.
A striking feature of carbohydrate metabolism in all parasitic
helminths 1s the partial breakdown of glucose, even under aerobic
conditions. With reference to the filariae, except for L.carinii, all
others studied so far i.e. B.pahangi, D.vlteae. Dirofllaria uniformis,
Chandlerella hawkingi, D.immitis and Setaria cervi, seem to be homolactate
fermenters (83,91,92). L.carinii converted 80% of the carbohydrate
utilized to lactic acid anaerobically, acetic acid production accounting
for the remainder (93). The generation of these acidic substances had
been alluded to previously by Taylor (94) and Earl (95); both investigators
reported a drop in pH of the media in which adult L.carinii and D.immitis
were maintained. Aerobically about one-half of the glucose consumed was
converted to lactic acid the rest to acetic acid and possibly glycogen
(83,93). Recently Rew and Saz (96) reported that microfilariae of
L.carinii. D.viteae and B.pahangi also metabolized glucose aerobically
to acetate and CO 2 . In contrast to the adults all three species of

21
mi cro Filari ids have an aerobic requirement for m otility but possibly
not for s u r v i v a l .
The principal pathway of glucose me ta bol is m in filariae is phosphory-
lative glycolysis down to p h o s ph o en ol py r uv at e , the steps being similar
to those in vertebrate tissues (97-99). Subsequently, in adults, relatively
high pyruvate kinase activity favours the production of pyruvate associated
with a net production of ATP. Pyruvate in turn is reduced to lactate in
the presence of lactate dehydrogenase. Filarial ph os ph o en o lpyruvate may
also be metabolized to oxaloacetate by way of CO^ fixation catalyzed by
phosphoenolpyruvate carboxykinase (97,100). Apparently, however, the
pyruvate kinase-mediated pathway seems to be favoured in most filariae
(100). Although an oxidative pathway to produce NADPH and ribose is
found in filariae (98,101) at present there is insufficient evidence to
postulate a complete hexose monophosphate shunt. NADPH is usually
linked with membrane stabilization (in which reduced gl utathione plays a
prominent role) and fatty acid synthesis. It seems unlikely that the
latter metabolic role operates in filariae, since nematodes apparently
rely on exogenous fatty acids.
Notwithstanding the detection of the tric ar bo xy lic acid (TCA) cycle
enzymes in adult filariae (102,103), it has been speculated that the low
levels of aconitase and isocitrate dehydrogenase might relegate the T CA
cycle to an insignificant position in the overall met ab ol ism of adult
filariae. Oya et aj_. (104) have intimated that the TCA cycle in helminth
parasites might be concerned with transamination reactions; buttressing
this notion is the detection in some helminths of glutamate dehydrogenase
activity which generates a - k e t og l ut a ra te . On the other hand it has been
reported in microfilariae that the TCA cycle functions, but at a very

22
low level of turnover (96). In addition to completely oxidizing glucose,
microf ilariae share with adult L.carini i the metabolic peculiarity of
oxidatively decarboxyl ating pyruvate to acetate (83,96,103). This
reaction might explain the oxygen dependence and generation of energy
for microfilaria! motility. It also might account for the aerobic
dependency of adult L . ca r in ii . As a consequence of fluoroacetate
inhibition it has been suggested that a fraction of the aerobically
produced C 0o of this filaria comes from the complete oxidation of
glucose (93). However, the activity of cytochrome c oxidase has not
been detected in suspensions of L .c a r i n i i (83,93).
Fractionation of filarial lipids revealed that phospholipids are
predominant, the major phospholipids being phosphatidylethanolamine, and
phosphatid y 1choline; phosphatidyl i no s i t o l , ca r di o lipin, sphingomyelin
and lysolecithin were minor components (105-107). Other classes of
lipids found in filariae were di- and tr i- a cy l gl yc e ro l s, free cholesterol,
cholesterol esters, fatty acids (106,108), ubiauinone(s) and short-chain
isoprenoid alcohol(s) (109). One group of investigators reported that
the composition of filarial fatty acids which was similar in all the
species examined, differed significantly from that of the host (106).
In contradiction, Warren and Daugherty (110) found that the lipid composition
of parasitic helminths was variable and depended on the host species.
Phospholipid synthesis has not y et been shown explicitly in any
filari id, even though it has been established in other helminth paracites
(111). The recovery of radioactivity from the lipid fraction of D.imniitis
microfilariae incubated with [U-"54C]-gl ucose at least implies that
filariae can synthesize phospholipids by way of glycerol (101).

23
Lxcept for Ascaris liimbricoi 2 S_ which seems to possess the ability
to manufacture fatty acids by way of the malonyl CoA pathway, all other
helminths including filariae appear unable to synthesize fatty acids
de n o v o , but instead are capable of lengthening preformed fatty acids by
condensation with acetyl CoA (108,111).
Although it is generally believed that parasites lack the necessary
pathways to synthesize sterol (111), some trypanosomes an^ filariae
have been reported to synthesize cholesterol de novo (108,111). On the
other hand, Comley e_t aj_. (109) were unable to detect radiolabelled
squalene and cholesterol in extracts of adult B.pahangi and D.imnitis
that were incubated in the presence of [ 2 - ^ C ] m e v a l o n a t e . These latter
investigators found that substantial radioactivity derived from [2-
^ C ] m e v a l o n a t e was recovered in short-chain isoprenoid alcohol(s) which
on TLC ran close to cholesterol; they suggested that the difference
between them and Turner and H u t c h i s o n (108) w i th regard to conclusions about
the cholesterol-synthesizing capacity of filariae might be due to the
method used by the two groups to separate cholesterol. In contrast to
their apparent inability to synthesize cholesterol, adult S.pahanqi and
D.immitis were able to synthesize radiolabelled ubiquinone 9 when they
were incubated with [2-^ C j me v a l o n a t e . Ubiquinone 9 had earlier been identified
in the lung worm M etastrongylus elongatus (112). The presence of
ubiquinone and rhodoquinone have been reported in a number of helminth
parasites (113). Although ubiquinone may substitute for menadione in
0 . imrni ti s N ^, N^ -m eth y le ne - te t ra hy d ro f ol ate (methyleneTHF) reductase
assay (109), it is probable that its presence in filariae indicates a
role similar to its participation in other eukaryotic and prokaryotic

24
mitochondrial electron transport. Interestin g l y , u l t r a s tructural
studies have shown that adult f i 1 r i ids have a large number of highly
cristated, well developed mitochondria normally associated with tissues
capable of oxidative phosphorylation (103). The possibility exists that
these highly structured mitochondria in filariae may function in an
electron transport-linked phosphorylation of ADP coupled to a pyruvate
dehydrogenase complex.
Lipid oxidation has been demonstrated both in A . 1umbricoides eggs
and free-living stages of other parasitic nematodes (111). This is not
surprising since these developmental stages have functional 3-oxidation
and TCA cycles. Even though Subrahmanyam (105) suggested that t r iglycerides
may be important energy reservoirs in L . c a r i n i i , it is u nlikely that the
oxidation of the acyl moieties of glycerides would account for this cs
the complete 6-oxidation and TCA-cycle enzymes apparently are absent in
adult parasitic nematodes (111); degradation of glycerol via glycolysis
seems more likely (114).
Turning to other areas of filarial metabolism, Jaffe and Doremus
(101) found that D.immitis m icr ofilariae readily incorporated adenine,
adenosine, uridine, and uracil as well as orotic acid into RNA but not
into DNA. Utilization of preformed thymidine was not detected. Identical
findings were reported by Chen and Howells (115) who studied adult
B.pahangi and by Simpson and Lawrence (116) who studied Brugia patei
laevae developing in A ed e s t o g o i . De novo synthesis of thymidylate
seems probable as thymidylate synthase activity has been detected in
filariae (117). It is also likely that filariae can synthe:>i■

>, u n d y l i c
acid from formation and decarboxyl a tion of orotidylic acid (111).
Although de novo synthesis of purine nucleotides by D.immitis mi cr ofilariae

25
could not be detected (101), more; recent investigations by Jaffe a.id
Chrin (117) have demonstrated thaL adult filariae have this capacity,
using 10-formylTHF and 5,10-methenylTHF to donate, res pe ct iv e l y , carbons 2
and 8 of the purine ring. Wong and Ko (118) earlier reported that the
parasitic nematode Angiostrongylus cantonensis can synthesise purine
ribonucleotide de n o v o . Adult B .pahanqi and D .immitis ap parently cannot
synthesise folic acid de n o v o ; instead, they appear to rely on a source
of preformed folate derived from their hosts. These filariae can oxidise
5-methylTHF directly to 5 , 10-methyleneTHF which in turn is connected to
other THF cofactors that participate in single-carbon donors as in the
enzymatic synthesis of serine from g ly ci ne an d thyrnidylate from d e o x y u r i d y l a te
in addition to the synthesis de novo of purine nucleotides (53). It is
noteworthy that 5-methylTHF is the major form of folate present in the
extracellular fluids that envelopeand nourish filariae, at least in
their vertebrate hosts (58). There is evidence that a severe d efi ciency
state is harmful to adult filariae in vitro (58). Deprivation of
vitamins A, E and B6 also has an adverse effect on filariae; for instance,
vitamin B5 deficiency of albino rats caused a state of a m ic r of i1a rein ia
in L.carinii (119). On the other hand thiamine deficient hosts were
more susceptible to infection with filariae (120).
As relatively little is known about filarial met ab ol is m and physiology,
a scientific working group on filariasis convened by the World Health
Organization Special Programme for Research and Training in Tropical
Diseases recommended supportive investigations into this area of the
parasites. It was the belief of the panelists that studies of this
nature could reveal hitherto unrecognized aspects of the filarial biology
and chemistry that could lead to the synthesis of selective antifilarial

26
drugs. Accumulated experience in related fields strongly suggests that
rational development of drugs is possible. It has been established in
the area of anti-infective c hemotherapy that clinically useful drugs
exploit qualitative or quantitative differences between parasites and
their hosts in some analogous metabolic or physiological process. The
traditional approach to a n t i-infective drug development, however, has
involved empirical screening (121) in which substances from a wide
variety of sources are tested for general growth inhibitory activity;
promising compounds are further tested to determine their c hemotherapeutic
potential.
With respect to the background for the specific biochemical studies
of filariae that are the subject of this thesis, Jaffe and colleagues
(122-127) delineated the pathways of folate-related m et a bo lis m in a
f i laria-susceptible strain of Aedes aeqypti mosquitoes and that a
characteristic change occurred in the activity of certain f ol a te -re lated
enzymes when the mosquitoes became infected with the parasites. The
activities of dihydrofolate reductase, serine h yd ro x ym e t h y l t r a n s f e r a s e ,

5 10
N ,N -methyleneTHF reductase and methionine synthetase mar ke dl y increased,
whereas that of N ^ - f o r m y l THFsynthetase c or respondingly decreased. This
pattern of change suggested that the folate me tab ol is m in infected

• • 5
mosquitoes was being altered to favour the synthesis of N -methylTHF
which is required for methionine synthesis (126). Jaffe and Chrin (123)
therefore hypothesized that the developing filarial larvae mi gh t be
depleting the arthropod host stores of methionine or N 5-methylTHF, or
both, thereby shifting the equilibrium of the folate coenzymes towards
the synthesis of either or both of these compounds.

27
Against this background it was proposed to investigate the
metabolism of methionine in filariae; delineating the metabolic
pathways, identifying the enzymes subserving the various steps
and characterizing the key enzymes. It was expected that
knowledge emerging from these studies would deepen our
understanding of the metabolism of filarial Darasites. It miqht
also help to assess the suitability of methionine metabolism as
a potentially exploitable target for antifilarial chemotherapy.

Cli -pter 2
MATERIALS AND METHODS
MATERIALS
[8 - ^ C ] A d e n o s i n e (sp. radioactivity 57.7 mCi/mmol), S-adenosyl-L-
[CH 3- 14 C]methionine (62 mCi/rnniol), L-[ 35 S]methionine (303 mCi/mmol), L-
[CH 3- 14 C]rnethionine (57.2 m C i / m m o l ) , [CH 3 - ] 4C]chol ine (59 mCi/mmol) and
[CH^-^ 4 C]-N^-methyltetrahydrofolate (58 mCi/mmole) were obtained from
Amersham Corp. Arlington Heights, IL. Minimum essential me dium Eagle
(MEME) with Earle's balanced salts (BSS) was from Microbiological
Associates, Bethesda, MD. Thin layer chromatographic plates were
purchased from Uniplate; Analtech Inc., Newark, DE. Suramin was kindly
provided by Dr. G. Lammler, Justus Liebig Univ., Giessen, Fed. Repub.
of Germany; diethylcarbamazine was from Lederle L a b o r a t o r i e s , Pearl River,
MY. L-Homocysteine was prepared from L-homocysteine thiolactone as
described by Mudd et aj_. (128). Molecular weight calibration kit was
purchased from Pharmacia Fine Chemicals, Inc., Piscataway, NJ. Unless
otherwise indicated all other chemicals were from Sigma Chemical Co.,
St. Louis, MO., and they were analytical grade.
Dogs infected with D.i mmitis and gerbils with B .pahangi were from
Dr. John W. McCall, Univ. of Georgia, Athens, GA.
METHODS
A. Delineation of Methionine Metabolic Pathway in F i l a r i a e : Adult
female B. pahangi were removed from the peritoneal cavities of the
gerbils, washed in 0.9% saline solution, blotted carefully, and groups
of two were placed in 2 ml of sterilized MEME and BSS. To each of five
28
29
such vials was added 15.16 uCi [v‘S]methionine, and the worms were
incubated at 37°C for 24 h in a tiiermosta ti ca 1 ly controlled water bath.
Thereafter, the worms v/ere washed quickly with 100 ml of 0.92 saline
solution, blotted dry, weighed and homogenised in a mini Potter -E lvehjem
homogeniser with 0.5 ml of 1.5Mperchloric acid for 1 h. A modified
version of the method of Shapiro and Ehninger (129) was used to process
the worms. The homogenate was centrifuged at 30,000 xg for 30 min in a

05 f
Sorval RC-2B refrigerated centrifuge. Metabolites of SJniethionine
present in the supernatant fraction were separated with the appropriate
carriers by thin layer chromotography (TLC) on silica gel F plates using
n-butyl alcohol-acetic acid-water (12:3:5 v/v/v) as solvent. AdoMet and
AdoHcy were detected under UV light (at 254) and the others by spraying
the chromatoplate with 0.22 ninhydrin in n-butyl alcohol and heating in
an oven at 110°C for about 10 min. Spots that co-chromatograpned with
standards were scraped off, and the radioactivity therein was determined.
B. Preparation of E xt r ac t: Adult D.immitis were quickly removed from
the heart and the pulmonary artery of a euthanised infected dog and
were placed in phosphate-buffered isotonic sal ine (pH 7.0)containinq 0. 5% glucose;
the worms were then repeatedly washed in this saline solution to remove
blood clots. They were sexed, blotted and either immediately processed
for enzyme assay or transferred into the appropriate medium for in vitro
experiments.
For enzymatic studies the worms were minced with scissors and
homogenised at 0°C in a P ot te r- El vehjem houogcniser in 4 ml cf
0.01M Tris-HCl ,buffer (pH 7,4) containing 0.25M sucrose, 0.01M MgCl 2
30
and 0.001M 2-mercaptoethanol (13(1) per gram of worm. The resulting
homogenate, to which fine glass b ads (3 parts of hornogenate:! part of
glass beads) were added, was sonicated at -12°C for four 20 sec intervals
at a power output of 60W using a Sonicator Cell Disrupter, Model W-220F
(Heat Systems-Ultrasonics, Inc, Plainview, Long Island, NY). The sonication
step was deleted in studies dealing with intracellular enzyme localisation.
The final homogenate was spun for 30 min at 30,000 xg at 2°C. The
supernatant fraction was dialysed overnight against the same buffer but
without sucrose at 4°C. Unless needed for immediate enzyme assay, the
dialyzed extract was stored at -70°C.
C. Enzyme A s s a y s :
(a) N^-Methy1THF:homocysteine S-methyltransferase (EC 2 . 1 . 1 . 1 3 ) : The assay
was according to the method of Mudd et^ aj_. (131) as modified by Jaffe
and Chrin (126).
(b) Betaine:homocysteine S-methyltransferase (EC 2. 1. 1. 5 ) : The method
employed was as described by Jaffe and Chrin (126).
(c) ATP:methionine S-adenosyltransferase (EC 2 . 5 . 1 . 5 ) : The assay
procedure was a modification of the method of Chiang and Cantoni (132).
The incubation mixture contained 84 mM of Tris-HCl, pH 7.8; 40 m M of
KC1; 20 mM of M gC l 2 ; 1 mM of KCN; 10 mM of ATP; 5 mfi of unlabelled
methionine; 50 nCi of [ CH ^- ^C ji n et h io ni n e; 1 m M of 2-mercaptoethanol ;
100 ul of extract and water to a final volume of 250 ul. The reaction
mixture was incubated at 30°C for 60 min and the reaction was terminated
by adding 0.05 ml of 40% (v/v) trichloroacetic acid. After allowing to
sit on ice for 10 min the precipitate was spun down. Twenty ul of the
31
supernatant fraction was applied on to precoated silica gel G TLC
plates (250 um). The c h r o m a t o g r a w a s developed in 1-butanol-water-acetic
acid (12:5:3 v/v/v). On spraying the plate with ninhydrin the spot
that co-chromatographed with authentic AdoMet was scraped off and counted
for radioactivit y .
(d ) S-Adenosylmethionine ( A d o M e t ) :homocystei ne S-methyltransferase
(EC 2 . 1 . 1 .10): The method of Mudd (134) was modified and was used
to assay the transmethylase. Contained in a total volume of 250 ul
were 0.1M potassium phosphate buffer, pH 7.5; 2 mM homocysteine; 62.5 nCi
S - a d e n o s y l - [ CH^ -^C jm eth ion ine and 100 ul of extract. The mixture was
incubated at 37°C for 60 min and the reaction was stopped with 50 ul of
1.84M perchloric acid. The subsequent treatment was as givc-n
in the procedure for ATP:methionine S-a den o s y l t r a n s f e r a s e . The
methionine spot was scraped off and counted for radioactivity.
(e) S-Adenosylhomocysteine (AdoHc.y)hydrolase (EC 3 . 3 . 1 . 1 ) : The enzyme
was assayed in the reverse direction by following the formation of
S-adenosylhomocysteine. The assay system contained the following:
0.05M potassium phosphate buffer, pH 7.0; 0.3 mM homocysteine; 0.4 nM
unlabelled adenosine; 50 nCi [ 8 - ^ C ] a d e n o s i n e ; 3 mM mercaptoethanol and
100 ul of extract. The total volume of the reaction mixture was 250 ul.
Incubation was carried out at 37°C for 30 min after which the reaction
was stopped with 0.05 ml cold 1.84M perchloric acid. Later treatment
was as described above except that silica gel GF. was used in place of
silica gel G. The spot containing AdoHcy was detected under UV linht,
scraped off and radioactivity therein determined.

(f) Cystathionine-B-svnthase (Ei 4 . 2 . 1 . 2 2 ) : The reaction mixture
consisted of the following: 40 mi'J potassium phosphate buffer, pH 8.0;
8 mM hom oc ys te in e; 2.5 m M serine; 0.12 m M pyridoxal phosphate; 100 ul of
extract and water to a final volume of 0.3 ml. After 60 min incubation
at 37°C the reaction was stopped by the addition of 0.7 ml ninhydrin
reagent which was composed of 150 mg ninhydrin, 15 ml glacial acetic
acid and 5 ml phosphoric acid (133). The colour was developed by heating
the reaction mixture at 100°C for 5 min; the tubes were then cooled on
ice and the absorbance at 455 nm read after 10 min against a m i n u s -
homocysteine blank.
(g) Cystathionine- % -lyase (EC 4 . 2 . 1 . 1 5 ) : A total assay volume of
0.5 ml contained 40 mM phosphate buffer, pH 7.5; 0.12 m M pyridoxal
phosphate; 2 m M cystathionine; and 100 ul of extract. The mixture ‘

.-as
incubated for GO min at 37°C. The reaction was terminated by rapid
cooling on ice for 5 min after which 0.5 ml of 10 m M dithiothreitol and
10 ul of 2M NaOH (134) were added. The reaction was allowed to continue
at room temperature for 30 min. Afterwards 2 ml of freshly prepared
ninhydrin reagent (133) was- added and the tubes were heated for 5 min at
100°C. After allowing to cool on ice for 2 min the absorbance at 550 nm
was read against a minus-extract blank.
All spectro ph ot om et ric readings were conducted using a Beckman
Model 25 Recording Spectrophotometer. Radioactivity was assayed by
means of liquid scintillation counting of samples in a toluene-based
cocktail of composition 6 g 2 ,5-diphenyloxazole (PRO), 500 m! absolute
ethanol and 1400 ml toluene using Packard Tri-Carb spectrometer Model
3002. Interna] standardiz a tion was used to correct samples for quenching.
33
Protein concentration was e s ; m a t e d according to the method of
Lowry e t a l . (135) with bovine se am albumin as standard.
Enzyme activity was expressed as micromole of product formed/hour/mg
protein under the particular assay condition. The amount of cystathionine
and cysteine formed was found by extrapolation from calibration curves.
D. Metabolic Fate of AdoMet in F i l a r i a e : To determine the fate of
AdoMet, two groups of 5 female D.immitis were placed in 50 ml of the
standard medium, containing 350 units/ml s t r e p t o m y c i n , to which was
added 25 uCi of L - f C H ^ - ^ C j m e t h i o n i n e . After incubation at 37°C for 6 h
the worms were removed, washed with six 50 ml aliquots of isotonic
saline, blotted, weighed and stored at -70°C until required for analysis.
(a) Extraction of P r ot e i n : The acid soluble fraction was obtained by
homogenising the worms twice with 1.5 ml of 1M perchloric acid at 0°C
and centrifuging the homogenate at 30,000 xa Tor 30 min at 2°C. The
pellet was washed three times with the same acid. Protein was extracted
by the method of Ogur and Rosen (136). To the protein fraction was
added an equal volume of methanol and the sample rotary-evaporated to
dryness- The residue was resuspended in 4 ml of methanol and again
dried. The dried sample was hydrolyzed in 2 ml of 6M HC1 for 40 hr at
110°C. The hydrolysate was dried under reduced pressure and the residue
was dissolved in 1.0 ml of deionised water. Methylated amino acids were
separated by ascending paper chromatography on Whatman #1 filter paper
with pyridine-acetone-3M NHÔH (50:30:25 by vol.) (137). The areas
corresponding to methionine, N - C - m e t h yl l ys in e , 3-methyl histidine and DL
a-methylhistidine were located by ninhydrin and the radioactivity therein
was determined.
34
(b) Extraction of Nucleic A cids: DNA and RNA were extracted essentially
according to the procedure of Huberman and Sachs (138). One hundred and
twenty-five milligrams of adult D.immitis were homogenised with 4 ml; of 0.?t)lM
Tris-HCl buffer, pH 7.4>containinn 0.01M EDTA, 0.01M NaCl and 1% sodium
dodecyl sulphate. The resulting homogenate was extracted with equal volume
of phenol, and the emulsion obtained centrifuged at 1,200 xg for 5 min.
The upper aqueous phase was extracted with an equal volume of phenol-
chloroform (1:1 v/v) and the nucleic acids precipitated overnight at -20°C
with 2 volumes of absolute ethanol after addition of 0.2M NaCl. After
spinning at 2,300 xg for 45 min. the precipitate obtained was dissolved
in water and 2M LiCl and the RNA precipitated overnight at 4°C. The
precipitate collected at 17,000 xg was dissolved in 0.05M Tris-HCl(pH 6.7),
25mM KC1 and 2.5mM l^qCl^ (TKto). The DNA remaining in the LiCl was
precipitated overnight at -2C°C with 2 volumes of absolute ethanol,
centrifuged at 12,000 xg for 30 minutes and dissolved in 1.5mM NaCl and
0.15mM sodium citrate, pH 7,2 (SSC). The RNA and DN'A solutions were
incubated for 90 minutes at room temperature with 50ug/ml DNase in TKM and
RNase 1n SSC respectively. Afterwards the solutions were deproteinized
by consecutive extractions with equal volumes of chloroform-phenol (1:1 v/v)
and chloroform. The nucleic acids were precipitated overnight at -20°C
with 2 volumes of absolute ethanol after addition of 0.2M NaCl and then
dissolved in 1.0ml of SSC. The absorbance at 260nm was taken against a
solvent blank.
The Isolated DNA and RNA were Incubated at 37°C for 1 hour with
0.1ml of 50 ug/ml DNase, RNase or prcnase to remove any further contaminating
nucleic acids and proteins. The radioactivities of the recovered
acid-soluble DNA and RNA after denaturation with 0.?ml 10% TCA in the
presence of 0.1ml 0.5% bovine serum albumin were determined.

35
(c) Extraction of Lipids: While still frozen, a batch of filariae
was manually crushed in a qlass homogeniser, and then total lipids were
extracted twice with each of the following solvents in the order given:
a) 100% methanol; b) 60% methanol:40% chloroform, and c) 40% methanol:60&
chloroform. Extractions were carried out at 0°C, each successive extract
was centrifuged under refrigeration at 1000 xg for 10 min, the pellets
were reextracted, all supernatant fractions were pooled, and finally
evaporated to dryness under reduced pressure. Phospholipids were eluted
with methanol from a column of silicic acid (100-300 mesh) and celite
(diatomaceous earth) (2:1 w / w ) , following the method of Burt et a l .(139).
The eluted phospholipids were rotary evaporated to dryness, resuspended

(100 ul)
in cyclohexane and a known aliquot/removed for measurement of radioactivity.
The phospholipid fraction was analyzed by thin layer chromotography on
silica gel H wtth chioroform-methanol-acetic acid-water (25:15:4:2 v/v/v/v)
(140) and with 5 mg/ml of the following dissolved in methanol-chloroform
(1:2 v/v) as standards: synthetic lecithin, lysolecithin, sphingomyelin,
dimethyl phosphatidylethanol amine and phosphatidyl ethanol amine. The
lipids were visualized by exposing the TLC plate to iodine vapour.
The TLC plate was then divided into 1-cm squares, spots scraped off and
the radioactivity in each determined. Another TLC plate was developed,
photographed and an autoradiogram prepared.
Autoradiography: Upon the disappearance of the iodine the chromatogram
was placed 1n opposition to a sheet of Kodak X-Omat-RP film (Eastman
Kodak, Co., Rochester, NY) and left in a light proof x-ray cassette
holder in the dark. At the end of a 2-week exposure period the film
was developed.
36
Chapter 3
RESULTS
A. Biosynthesis of Methionine by Filariae
Experimental findings indicated that at least adult filariae are
unable to synthesise methionine de novo and instead probably rely upon an
exogenous source of this amino acid. Repeated attempts to detect the
presence of vitamin B12-dependent and independent methionine synthesising
enzymes proved fruitless (Table 3), Moreover, incubation of adult
B.pahangi with N 5 -(CH3- 14 C)methylTHF ( 14 CH 3 THF) revealed that there was no
incorporation of radioactivity into methionine, as compared with the
substantial counts in methionine extracted from mammalian cells (L 1210)
incubated under identical conditions (Table 1). The results confirm those
reported by Jaffe et^ al (142).
Table 1: Biosynthesis of Methionine by Filariae
Female B.pahangi were incubated with 20 uCi of ^ C H 3T H F for 2 and 24 h.
At the end of the incubation period the worms were washed with 0.9% saline
and processed by the procedure of Schmidt and Thannhauser (141). The
Trichloroacetic acid (TCA)-soluble fraction was analyzed chromatographically
for methionine by the method of Jaffe and Chrin (126). Incubation of L 1210
under identical conditions was undertaken for comparison.
COUNTS/MINUTE
SYSTEM 2 h 24 h
B.pahanqi 0 0
L 1210 1526 4130

37
B. Ability of Filarial Worms to Metabolize Methionine
To test for the ability of filarial worms to metabolize methionine

or
adult B.pahangi were incubated in vitro in the presence of (JJS) methionine.
The results are given in Table 2.
Table 2: Ability of Filarial Worms to Metabolize Methionine

oc
Adult female B.pahangi were incubated in the presence of ( S) methionine.
After 24 h the worms were homogenized in perchloric acid and the supernatant
fraction obtained analyzed chromatographically for metabolites of methionine.
Details of experimental procedure are given in Materials and Methods section.
cpm/mg worm
Methionine 149
S-Adenosylmethioni ne 651
S-Adenosylhomocys tei ne 792
Homocysteine & cysteine 388
There was incorporation of radioactivity into AdoMet, AdoHcy, homocysteine
and cysteine, demonstrating that filariae possess the enzymatic machinery
for metabolizing methionine analogous to that found in prokaryotes and
other eukaryotes. The enzymes involved were subsequently detected in
filarial extracts and their specific activities are shown in Table 3.
Prior to determining specific activities linearity was established between
enzyme activity, duration of assay, and protein concentration. Relatively
high levels of S-adenosylhomocysteine hydrolase and the transsulphuration
enzymes were found, though generally the activity of the methionine
metabolizing enzymes in filariae under these experimental conditions was low.

38
Table 3: Specific Activities of Some Filarial Methionine Metabolizing Enzymes
Dialysed supernatant fraction of freshly obtained female D.inmltis
was used 1n the various enzyme assays. For details on extract preparation
and assay systems, see the section on Materials and Methods.
Enzyme Specific A c t i v i t y ^
ATP:meth1onine S-adenosyltransferase 0.02
S-Adenosylmethionine:homocysteine S-methyltransferase 0.11
S-Adenosylhomocysteine hydrolase 5
Cystathlonlne-jj-synthase 225
Cystathlonine-tf-lyase 7
Methionine synthetase 0
Beta1ne:homocysteine S-methyltransferase 0
^ n m o l e product formed/h /mg protein
C. Partial Characterisation of S-Adenosylmethionine.‘

Homocysteine
S-Methyltransferase
Despite the absence of vitamin B12-dependent and -independent methionine
synthesis 1n filarial worms, the detection of AdoMet:homocysteine
S-methyltransferase in filarial extracts suggested that these parasites could
meet some of their methionine requirement by way of this enzymatic reaction.
An attempt was made to partially characterize this enzyme.
In preliminary experiments it was found that the activity of this enzyme
was slightly higher 1n supernatant fractions of extracts maintained at 50°C for
5 min; consequently this heat treatment was used to partially purify the enzyme.
A sumnary of the purification procedure is shown in Table 4. A five-fold
Increase in activity over the crude extract was achieved, with the yield
almost unchanged.
39
T a b l e 4: Partial Purification of Filarial AdoMet .-Homocysteine S-Methyl transferase
Purification Volume Total Acct. Protein Sp.Act. Yield Deg. of

Step (ml) (*) Cone. (units/mg ( %) Purification
(mg/ml) protein)
Crude extract 21 10.3 4.45 0.11 100 1
50°C for 5 min 15 9.7 1.25 0.52 94 5
*nmole of methionine produced per hour.
(a) Intracellular Localization of AdoMet:Homocysteine S-Methyltransferase
Differential centrifugation studies coupled with deoxycholate treatment
of the resulting pellets indicated that AdoMet:homocysteine S-methyltransferase
1s exclusively cytosolic (Table 5).
Table 5: Intracellular Localization of AdoMet:Homocysteine S-Methyltransferase
Homogenate of adult female D.inroitis was subjected to differential
centrifugation. The activity of the methyl transferase in the various
fractions was determined under optimum assay conditions.
Fraction d.p.m. recovered in methionine
Nuclear 0
Mitochondrial 0
Microsomal 0
Supernatant (cystosol) 1909
(b) Dependence of Enzyme Activity on Time and Enzyme Concentration:
The activity of AdoMet:homocysteine S-methyltransferase was linear
with time for up to 120 min, after an initial lag period of 30 min (F i g .5).
The rate of production of methionine was also directly proportional to
the amount of enzyme present in the incubation mixture (Fig. 6).

40
6
ro
O
CL
Cl
0 30 60 90 120
TIME (MIN.)
Figure 5
Time-Course of Formation of Methionine by
AdoMet:Homocysteine S-Methyltransferase
Different reaction mixtures were set up. At the end of the incubation
period the reaction was terminated with perchloric acid. The radiolabelled
methionine formed was separated chromatographically and estimated by liquid
scintillation spectrometry. Details of the procedure are found in the
Materials and Methods.

41
AMOUNT OF ENZYME (MG.)

Figure 6
Formation of Methionine by AdoMet:Homocysteine
S-Methyltransferase as a Function of Enzyme Amount
Different volumes of the enzyme preparation (2 mg/ml) w e re incubated
as described in the standard assay. The amount of product formed was
determined by liquid scintillation counting.

42
(c ) Effect of pH and Ionic Strength on Enzyme Activity: The pH-acttvity
profile in phosphate buffer gave a single sharp peak at pH 8.0 (Fig.7).
However, because of the instability of AdoMet in alkaline medium (195-198)
the effect of ionic strength was investigated at pH 7.5, varying the
concentration of the buffer from 25-200 mM. The activity of the enzyme
was optimal at around 100 mM (Fig. 8),
(d) Effect •of Temperature on Enzyme Activity: Fig. 9 shows the effect of
temperature on AdoMet:homocysteine S-methyltransferase, The activity of
the enzyme increased up to 50°C, after which it declined rapidly.
(e) Requirement of A s say: Table 6 indicates the requirements for the
filarial AdoMet:homocysteine S-methyltransferase reaction. No methionine
was produced when the extract was omitted. In the absence of homocysteine
a limited amount of methionine was formed, even after extensive dialysis.
Of a variety of thiol compounds tested in place of mercaptoethanol, cysteine
gave the highest rate of product formation; dithiothreitol (DTT) and
reduced glutathione (GSH) were slightly inhibitory.
Table 6: Requirement of Assay of Filarial AdoMetrHomocysteine S-Methyltransferase ^
The complete assay system is described in the Materials and Methods.
Nanomole methionine/h /ml
Complete assay mixture 0.228

Minus homocysteine 0.017
Minus mercaptoethanol 0.123
" " + cysteine 0.527
H " + dithiothreitol 0.201
" + reduced glutathione 0.156
" extract 0
^ E x t r a c t was prepared in 0.04M Tris-HCL buffer, pH 7.4

43
NMOLE M E T /H R /M L
pH
Figure 7
Effect of pH on A d o M e t :Homocysteine S-Methyltransferase
The methyl transferase assay was run in 0.1M potassium phosphate
buffer of varyinq pH values. The amount of labelled methionine
formed was determined as described in the standard assay.

44
MET/HR/ML
NMOLE
CONCN. OF BUFFER (mM)
Figure 8
Effect of Ionic Strength on A d o M e t :Homocysteine
S-Methyltransferase
The assay was performed in various concentrations of potassium
phosphate buffer pH 7.5. The rest of the procedure is as described
in the standard assay.

45
TEMPERATURE (°C)
Figure 9
Effect of Temperature on AdoMet:Homocysteine S-Methyltransferase
The enzyme assay was run at various temperatures under conditions
given in the standard method.

46
(f) Effect of Divalent Mst.al Ions and E D T A : All the metal ions studied
activated the methyl transferase (Table 7) The alkaline earth metal ions,
Mg++ and Ca+ + , were more effective than the transition divalent metals,
Mn+ + , Zn++ and Co+ + .
Table 7: Effect of Metal Ions on Filarial AdoMet: Homocysteine S-Methyl t r a n s f e r a s e ^
The enzyme was incubated with various divalent metal ions and the enzyme
activity was measured as in the standard assay conditions.
10“3M Cation Nanomole methionine formed/.h /ml
None 0.225
Mg++ 0.551
Ca++ 0.428
Mn++ 0.299
Zn++ 0.285
Co++ 0.320
As a result of the above findings the effect on the methyl transferase of E DT A
was investigated; EDTA concentrations greater than lCT^M were found to be
inhibitory (Table 8 ).
Table 8 : Effect of EDTA on Filarial AdoMet: Homocysteine S-Methyl t r a n s f e r a s e ^
The Enzyme was incubated with different concentrations of EDTA under
normal assay conditions.
EDTA Concn (M) Nanomole methionine formed/h /ml
None 0.396
10'5 0.450
10'4 0.263
10-3
0.186
47
(g) Kinetic Studies: The partially purified enzyme preparation
exhibited Michaelis-Menten kinetics with respect to both homocysteine
and AdoMet. The kinetic constants obtained by the double-reciprocal
plot method of Lineweaver and Burk C143) are found in Table 9.
Table 9: Kinetic Parameters of Filarial AdoMet: Homocysteine S-Methyltransferase
The values of the apparent Km and Vmax at pH 7.5 and 37°C were estimated
from Figures 10 and 11.
Substrate Vmax Km (uM)
AdoMet 0.59 8
Homocysteine 0.39 900
The value of Vmax is expressed as nmole methionine formed/hr/ml.
th) Effect of Inhibitors:
The effect of the standard antifilarial drugs, DEC and suramin,
on the activity of AdoMet: homocysteine S-methyltransferase was
investigated. DEC and suramin inhibited this filarial enzyme by 32%
and 67% respectively, at a concentration of 0.1 mM. At the same level
of AdoHcy there was no effect.

48
University of Ghana
http://ugspace.ug.edu.gh
49
was varied holding that of AdoMet constant at 0.016mM.

50
D. Metabolic Fate of AdoMet in Filariae: Because of the significance
of AdoMet-dependent methylatlons in cell function (144-146), identification
of other methyl acceptor compounds in filariae was undertaken. This was
preformed by incubating adult female O.immitis in a medium containing
(CH^-^C)methionine to induce these filariae to synthesise radiolabelled
AdoMet. The distribution of this radioactivity among the various filarial
fractions was as shown in Table 10.
Table 10: Distribution of Radioactivity after Incubating Adult D.inrnitis
in the Presence of CH^- ^ C ) Methionine
Adult female O.immitis were incubated in (CH3-^C)methionine for 6h.
Afterwards the parasites were processed and the radioactivity in the
various fractions determined by liquid scintillating counting. Details
of the experimental procedures are described in the Materials and
Methods.
Fraction dpm %
Acid-soluble 145136 42.4
Total lipids 71369 20.8
Phospholipids 68941 20.1
Neutral lipids 1000 0.3
Protein 126192 36.8
Nucleic acid 0 0
51
It can be seen that the total lipid fraction contained 21% of the label
incorporated by these parasites, most of the counts being found in the
phospholipid fraction. TLC of the filarial phospholipid fraction (Fig.12)
indicated that the radiolabelled phospholipids were dimethylphosphatidylethano-
lamine, lysolecithin, lecithin and sphingomyelin. The incorporation of
radioactivity into dimethyl phosphatidyl-ethanol amine was of the same amount as
that associated with the other phospholipids. An autoradiograph, however,
indicated the presence of sphingomyelin (Fig. 13). The other spot in the
autoradiograph was identified as methionine. This is because on another
TLC plate a spot with relative mobility close to that of the spot in the
autoradiograph had co-chromatographed with authentic methionine. In between
the methionine and sphingomyelin spots was a diffuse band of radioactivity.
Sphingomyelin as well as lecithin, lysolecithin, phosphatidylethanolamine
and phosphatidylinositol could be identified among the phospholipids
isolated from adult female D.immitis (Plate 1). The composition of the dog
heartworm phospholipids closely resembled that of Setaria cervi, a nematode
parasite of water buffalo, Bubalus bubal is (107).
The remaining radioactivity was associated with the protein and
perchloric acid soluble fractions; the former accounted for 37% of the
total dpm incorporated. Fractionation of the protein hydrolysate indicated
radioactivity in methionine and methylated lysine and histidine, and some
unknown compounds which ran close to the origin (Fig.14). Methylated arginine
might be one of these unknowns as there was a spot which cochromatographed
with authentic arginine.
In the present investigation no radioactivity was recovered from the
nucleic acid fraction during the six hour period of incubation.

Figure 12
Adult female D.immitis were incubated in ( C H ^ - ^ C j m e t h i o n i n e for 6 h.
Afterwards the phospholipids were extracted and characterized
chromatographically. Details of the experimental procedure are
described in Materials and Methods.

52
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Figure 13
Autoradiogram of Filarial Phospholipids
(See page 54 for composition of 9 and 10)
autoradiograph prepared from the chromatograph

54
PL. 1: Photograph of Iodine-Developed TLC of Filarial Phospholipids
Adult femalfi D.immitis were incubated for 6h In (CH^-^Cjmethionine.
Isolation of the phospholipid fraction was followed by Its resolution
by thin layer chromatography. The TLC plate was developed, photographed f
and later autoradiographed. Experimental details could be found in

Materials and Methods.
1. Lecithin
2. Lysolectthin
3. Sphingomyelin
4. Dimethyl phosphatidyl ethanol amine

5. Natural lecithin from egg yolk
6. Phosphatidyl inositol
7. Phosphatidyl ethanol amine

8. Unlabelled phospholipid fraction (cold)
9. Cold + labelled phospholipid fraction

10. Labelled phospholipid fraction + standards (1-7)
Figure 14
Radioactivity Distribution in Thin Layer Chromatogram of Protein Hydrolyzi
Protein fraction extracted from adult female D.immitis incubated for 6h
in (CH^-^Cjmethionine was hydrolyzed in acid. The hydrolyzate was
analyzed chromatooraDhically for methylated amino acids. Details have
been described in Materials and Methods.

56
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DISCUSSION AND CONCLUSION
A« Folate Metabolism
ILt role of folic acid in the do novo synthesis of purii.j ribo
nucleotides and the pyrimidine d e o x y r i b onu cle oti de , thynndylate, has
been a::.ply demonstrated in many biological systems including parasitic
nematodes ( 147,148). Jackson and Siddiqui (149) have shown that
tieoaplectana q l a s e r i , a parasitic nematode of insects9needs folic acid
for reproduction since transformation was halted in the presence of
aminopterin and methotrexate, potent inhibitors of dihydrofolate
reductase. In addition to the above physiological functions, folic acid
is also involved in the biosyntnesis of methionine (14?) and tne prevision
of the fonnyl group of u-fortty!n:ethicnyltransfer ribonucleic c~ic! (ibO).
In view of this, any discussion of Methionine n et abolism in filariae
would be incomplete without considering the role of folate f • *bol is.;: in
filariae. The interrelationship ht-jtween methionine and folate i sin
is shown in Fig. 15., Jaffe and associates (57,117) have showM that
adult filarial wo rats possess the whole array of folate-rekv.i-rf enzymes.
Apart from some minor differences, the properties of the eiuyn;LS a s s o c i a t e

l" 10 . . .
with i'i'VJ -nethylenetotrahydrofolate(CH^THF), namely serine l.ydroxyiircthyl-
trarisferase (EC 2.1.2.1), thynn’

dylate synthetase (F.C 2 .1.2.4b), CH^TliF
dehydrogenase (fc'C 1.5.1.5) and CH^THF reductase (fcC 1,1 - •.53) ari>
generally similar to the analogous en;:ymes from ua^*al ian i.sosquito
sources. (122,124,125). tinlike microorganisms when: the reeulotion f
CH^TIIF d e h y d r o g e n a s e a c t i v i t y a p p e a r s to c o n t r u l fol.ê :.:elhioninA
metabolism (ISO), in the vertebrate and invertebrate systems the reactions

'0
of regulatory interest are those catalyzed by Ci-WHlr reductase ana N' -
foruiylTHF dehydrogenase (KC 1.5.1.6} (151,152). Ihese enzy.Mtic steps
58
59
ensure regeneration of tetrahydrofolate which otherwise would have been
trapped. Methionine, probably through AdoMet, exerts a modulatory
influence on these enzymes. In contrast to its activatory effect on
formylTHF dehydrogenase (151) methionine causes a negative feedback
response on the corresponding reductase (153,154). Consequently on
saturation of the folate system with methylTHF any excess one-carbon
units will be channelled towards the formylTHF dehydrogenase step and
eventually dissipated in the form of CO^ (151). Regulation of folate
metaholism may also be achieved by controlling the uptake mechanisms and
the formation of polyglutamates from monoglutamates (151).
Filariae have a relatively highly active NADP-dependent and NADP-
independent 10-formylTHF dehydrogenase system compared with its counterpart
in mammalian organs such as the liver; the general properties of the
analogous filarial and mammalian 10-formylTHF dehydrogenases are similar (58).
B. Methionine Metabolism
(a) N^-Meth.ylTHF: Homocysteine S-Methyltransferase and B e t a i n e :Homocysteine
S-Methyltransferase:
In all biological systems examined so far the thermodynamics of
CH 2THF reductase has always favoured the synthesis of methylTHF which is
utilized in the formation of methionine (147).
Methionine synthetase which catalyzes this reaction has been isolated
from Escherichia coli K-12 and purified to homogeneity, yielding three
components (155). These are: M component, methionine synthetase with molecular
weight of 186,000 daltons and containing one mole of vitamin B12 per mole
of protein; R component, flavine adenine dinucleotide (FAD) flavoprotein with
molecular weight of 27,000 daltons; and finally the F component,
flavine mononucleotide (FMN) molecular weight 19,400 daltons.
The three components and S-adenosylmethionine are essential for the

biosynthesis of methionine. TheS'.' accessory factors are utilized to
activate the B12 prosthetic group, a process which involves the conversion
of the protein bound B12 to methyl-B12 with the simultaneous reduction
of coba 11 (1 1 )«j cob (11)a 1ami n to methyl cob(I)al ami n (1 56,1 57-1 59). The
reaction sequence was derived spe ct rop hotometrically; the spectrum of
the M component as isolated showed the cobalt of B12 to be in the +2
valence state. Addition of NADPH (NADH substituted for NADPH but the
concentration required was higher owing to its higher Km value) R and F
did not alter the spectrum. This is because the reduction of B 12 (+2) to
812(+1) is so highly endergonic (160). With the addition of S-adenosyl-
methionine, enough energy was generated to shift the spectrum to that of
methyl-B12 (161,162).
5
Methylation of the activated methionine synthetase by !J -ne thy ltetra-
c
hydrofolic acid (N^-methylTHF) appears to be an intermediate in the
synthesis of methionine (163,164). This view was subsequently confirmed
(165-167). The methyl group is then donated to homocysteine to yield
methionine. Current studies apparently suggest that N -methylTHF poly-

c
glutamates are better substrates than -methyl-THF (the monoglutamate
form) (168,169). There is also evidence that the same enzyme ;r.ay be
mediating the mono- and poly-glutamate-utilizing reactions.
Methionine biosynthesis in mammals, unlike that in bacteria, requires
in addition to S-adenosylmethionine, R, F and M components, rranscobalamin
II, a serum protein for transporting B12 across the cell membrane (170
172). Once inside the cells B12 is converted to adenosyl 812 and m e thy l81
the former predominating (173).

61
As indicated in Tables 1 and 3 adult, filariae appear to be incapable
of methionine synthesis from homocysteine even though they possess
CH^THF reductase. Instead they have been found to assimilate methylTHF
and oxidise this cofactor to CH^THF by way of CH^THF reductase operating
in the reverse direction (117), This uncommon property has also been
observed in marnnalian foetal brain where ^C-labelled CH^THF was incorporated
into the DNA's thymine (144). This metabolic peculiarity suggests that
when methyl group synthesis and therefore involvement of folate in AdoMet
metabolism is insignificant most of the methionine would be derived
exogenously (144). It has been shown that the synthesis of
tetrahydroisoquinoline also involves the reversal of the reductase step (144).
The formaldehyde generated by this reaction condenses with dopamine in a
Pictet-Spengler reaction. Recently Jaffe and Chrin (117) employed this
unusual feature to illustrate the ability of adult filariae to synthesize
purines de novo.
Du Vigneaud and coworkers (174) observed that in the presence of
sufficient dietary choline, folic acid, vitamin G12, serine and
glycine, utilization of preformed methyl groups was preferred to de novo-
synthesized methyl groups. This observation stimulated the search for
(an) alternative B12-independent pathway of methionine synthesis that
culminated with the report that in mammals methionine could also be
produced in the presence of homocysteine plus choline, betaine or certain
methylsulphonium compounds such as dimethylacetothetin, ethylmethylacetothetin
and dimethylpropiothetin (175). Thetin-homocysteine methylpherase catalyzed

62
this reaction. The preferred substrate for the enzyme, dimethy-lacetothetin,
has, however, not been detected in nature. A second transmethylating enzyme,
betainerhomocysteine methylpherase or transferase with a high affinity
for the naturally occurring methyl donor, betaine, was later discovered (176).
The livers of all the vertebrates investigated appeared to have a
high activity of this transferase (177). Structural-activity studies on
the transferase seem to suggest that, the carboxyl moiety of betaine is not
required for activity while the N-methyl groups are essential (178).
The molecular weight of the pig liver enzyme has been estimated to be
270,000 daltons by sedimentation equilibrium (179).
Contrary to its wide occurrence in vertebrates, betaine:homocysteine
S-methyl transferase has been r e p o r t e d to be present in only one invertebrate,
the pond mussel, Anodona c.yqnea (177). Filarial worms are no exception
to other invertebrates; methionine biosynthesis by this pathway could not
be detected in extracts of these parasites (Table 3).
The finding that filariae are unable to synthesise methionine is
consistent with the report of Tkachuck et aj_. (180) that B12-dependent
methyl transferase activity has not yet been detected in any parasitic helminth
whereas the enzyme is found in the free-living nematode Caenorhabditis brigqsae
(181). Parasitic nematodes therefore probably derive their methionine from
an external source (182) unlike mammals, where even though this amino acid
is considered "essential", they still have the capacity to synthesise a
limited amount of it by the two pathways. On the other hand, the ability
to synthesise methionine has been claimed to occur in certain protozoan
parasites. Langer et al^ (183) observed that 1n P.berghei, de novo
synthesis contributed 20% of the total amount of methionine. This
minimal level of biosynthesis has prompted Bueding to question whether
activation of proteolytic release of methionine by homocysteine might
not explain the data of Langer et a l . (183). Although methionine

63
formation is found in Crithidia fasciculata, it is by a novel pathway.
This kinetoplastic flagellate possesses a folate-dependent mechanism for
producing «-keto-hydroxybutyrate from phosphoenolpyruvate and the jj-carbon
of serine (184). In the presence of organic sulphur, methionine is formed.
The inability of adult filariae to synthesise methionine de novo
may be an adaptive feature, in which case the deficiency might be
expected to exist in all the developmental stages. This is suggested by
the pattern of change in folate-related enzymes 1n filaria-infected
mosquitoes. Jaffe et a (122,123,125-127) observed an elevation in the
activities of dihydrofclate reductase, serine hydroxymethyltransferase,
CH^THF reductase and methionine synthetase whereas the level of
10-fornylTHF synthetase correspondingly declined. It was suggested that
the developing filarial larvae might be depleting the mosquitoes of their
methionine or N^-methylTHF stores or both. This picture may, however, be
different in helminth parasites with free-living stages. In these helminths
it is possible that some of the enzymes apparently absent or inactive in the
parasitic adults may in fact exist and function in the free-living
developmental stages.
(b) ATP: Methionine S-Adenosyltransferase
Although they differ from mammals by being unable to synthesise
methionine, filarial worms are similar to mairmals in that they have the
enzymatic machinery to metabolize this amino acid as indicated by
Tables 2 and 3. Filariae also resemble Plasmodium knowlesi in this

OC
respect. P.knowlesi was demonstrated in vitro to convert (J‘
'S)methionine
into (J*/S)cystine (185), The levels of the enzymes catalyzing the
transformation of methionine to homocysteine and adenosine in filariae

64
are about two to three orders of magnitude less than the corresponding
ones in the rat liver; the levels of the transsulphuration enzymes are
between 1 to 10 times lower in activity than the analogous mammalian
enzymes (186,187).
Activation of methionine appears important in the further metabolism
of this amino acid. In a classical contribution, Cantoni (188) identified
the active form of methionine as being AdoMet; adenosylation of
L-methionine at the expense of ATP, catalyzed by AdoMet synthetase appears
to be the sole pathway for the formation of AdoMet (189). AdoMet
synthetase is ubiquitous in nature (190,191). The fragmentary studies
of the tissue distribution of AdoMet synthetase in mammals suggest that
the enzyme is most active in the liver, with females exhibiting a higher
activity than males (192-194).
Owing to the instability of AdoMet in alkaline media, the activity
of the enzyn.e has usually been determined around neutral pH (195-19.").
But where the ATP-Mg ratio has been optimized, an activity peak at
pH 9.0 has been reported (197). Below pH 7.8,Mn++ was found to be 33
effective as Mg+ + . In addition to divalent cations, AdoMet sj.nthetise

. + + +
also requires monovalent cations such as K , Rb , or for maximal
activity (197).
The limited information available on the nucleotide specificity of
AdoMet synthetase suggests that only ATP and to a very minor
extent UTP in mouse liver (199) are active. Uata on the specificic;. of
methionine show that only few modifications would be compatible with
enzyme activity (200). The enzyme requires a carboxyl group (or short
ester) and an amino group (or short ac.yl.\ j derivative) attached to a

65
carbon atom bearing a hydrogen atom and linked by a two carbon bridge to
an ether sulphur (or selenium) atom bearing a short alkyl substituent
(either methyl or ethyl). In the course of the formation of AdoMet
there has been observed product-activated tripolyphosphatase activity
(189,196,134,201-203) which has led to the suggestion that an enzyme-
bound tripolyphosphate might be an intermediate in the reaction mechanism.
Chiang and Cantonl (132) recently purified bakers1 yeast AdoMet
synthetase to homogeneity; two forms of the enzyme were demonstrated.
The apparent molecular weight for both forms of the enzyme as determined
by Sephadex 6-150 column chromatography was 110,000 daltons, in agreement
with a value of 100,000 daltons obtained by Green (197) using analytical
ultracentrifugation. Molecular weights of 150,000 and 45,000 daltons
have also been reported (196,204).
As indicated earlier, the level of ATP: methionine S-adenosyltransferase
in filariae is very low. This might possibly account for the unsuccessful
attempts at characterizing this enzyme. The low activity might in turn be
attributed to the state in which some of the worms were received. Some of
the worms were received surrounded by water, the ice having thawed.
(c) Metabolic Functions of S-Adenosylmethionine
Once synthesised, AdoMet is utilized in various reactions. It
participates in most biological methylations (Table 11) by donating its
methyl group to acceptor molecules. Not only is it a methyl donor, the
relatively high energy of AdoMet provides the energy for the largely
exergonic transmethylation reactions (200).

66
T/vi LE 11
Some Examples of Transmethylation Re ac ti o n s I n vo l vi n g A d o M e t
Acceptor Molecules P r o d uc t
N-Acetyl-5-hydroxytryptamine Melatonin
Carnosine Anseri ne
Epinepherine Metanephrine
Guanidinoacetate Creatine
Histamine N-Methylhistamine
Nicotinamide N'-Methyl nicotinamide
Norepinephrine Epi nephri ne
Phosphatidyl ethanol ami ne Phosphatidylcholine
Lysyl residues of proteins N-Methylated lysyl residues
Carboxyl groups of proteins Methyl esters
Purines Methylated purines

67
Lipids constitute an important chemical pool for the metabolism of
methyl groups in animals. Filariae appear to be no exception; twenty-one
percent of radioactivity of (CH^-^C)methionine were found in the lipid
fraction. The level of radioactivity incorporated into the total lipids
of filariae compares with that obtained in other systems. Studies have
shown that after a one hour incubation of Trypanosoma equipsrdum in
(CHg-^Cjmethionlne, 8% of the radioactivity were recovered from the
total lipid fraction (205), whilst 27-34% of the label were incorporated
into the total lipid fraction of two strains of Aerobacter aerogenes (206).
Almost all the radioactivity in the lipid fraction was found in the
phospholipid component. The recovery of radiolabelled phospholipids
clearly shows that the unlabelled phospholipids isolated from female
D.lmmttis (Plate 1) and other parasitic nematodes (102,199) do not
originate solely from the host but that these parasites also possess a
biosynthetic capacity for these compounds. The results further reveal
the existence 1n filariae and probably other parasites of a transfer of
methyl groups from methionine to phosphatidylethanolamine in a similar
way to the process described in vertebrates (207) and invertebrates (208).
This is a stepwise methylation reaction catalyzed by two microsomal
methyl transferases (209-211). Methyl transferase I (a Mg++ requiring enzyme)
has a high affinity for AdoMet (Km of mammalian enzyme 1.4 uM) and catalyzes
the formation of phosphatidylmonomethylethanolamine (PME). Methyl transferase II
mediates the conversion of PME through phosphatidyldimethylethanolamine (POE)
to phosphatidylcholine. It has a Km for AdoMet of 100 uM. This pathway is
estimated to contribute 20-40% of the total lecithin pool in mammals (212),
cytidylyldiphosphate-choline incorporation into a, p-diacylglycerate (213)

accounting for the remainder.
68
In addition to detecting radioactivity in lysolecithin and lecithin,
counts were also observed in sphingomyelin. But the formation of the
latter needs clarification. The terminal phosphocholine moiety of
sphingomyelin could be derived from phosphatidylcholine by the action of
phospholipase C or D or a nonspecific phosphatase. The labelled
phosphocholine or choline released could either interact with unlabelled
sphingomyelin in an exchange reaction or complex with ceramtde, the parent
compound of sphingolipids, in an energy dependent reaction.
In addition to the biosynthesis of phosphatidylcholine, trypanosomids
also possess the ability to incorporate methionine into their sterols.
Meyer and Holz (214) found that Crithidia fasciculata incorporated
(CH3-^C)methionine into ergosterol. The bloodstream and culture form of
Trypanosoma lewisi and culture form of T.rhodesiense also synthesized
some unidentified sterols from acetate and methionine (111).
The formation of fatty acid methylesters which has been reported in
several biological systems incubated with (CHj-^C)methionine (215) could
not be followed in the present study because of the inavailability of the
appropriate markers. Although the silicic acid-celite column would resolve
these substances from the phospholipids, inefficient separation could
explain the radioactivity at the solvent front since fatty acid methylesters
migrate ahead of the phospholipids in the development solvent employed in
this experiment. But there was not enough time to try other fractionation
systems to completely separate the phospholipids and the methylesters.
However, 1t has been observed from other studies that the presence of
the latter substances could be artifactual as extracts in methanol could
induce either enzymatic or chemical transesterification reactions (215).
The amount of radioactivity retrieved from the filarial protein is
similar to the level reported for A.aerogenes (206) and T.equiperdum (205),
69
The isolation of methylated lysine, histidine and arginine from filariae is
a further manifestation of the ubiquity of protein methylation (144).
Several amino acid residues are known to be methylated usually at the
nucleophllic atoms by specific methylases. N-methylation at lysyl,
argininyl and histldyl residues and O-methylatlon at free carboxyl groups
are catalyzed by specific AdoMet-dependent methyl transferases. These
covalent modifications of amino acid residues have been implicated among
the posttranslational mechanisms for controlling protein function (144).
£-N-Methylated lysine residues are known to occur in a wide variety
of proteins from many sources (216). Among these cytochrome c's from
fungi, plants and protozoans have been found to possess one or more of
this amino acid (216). In contrast, none of the cytochrome c's of
either vertebrates or invertebrates possess C-N-methylated lysine. The
C-N-trlmethyllysine of trypanosome cytochromes makes the pigments of the
parasites less basic than the vertebrate cytochromes (111). Methylation
of cytochrome C of yeast or Neurospora crassa has been shown to facilitate
the binding of the cytochrome with mitochrondria, and subsequently play an
important role in the process of electron transport (144). Although actin
and myosin also contain significant amounts of various methylated basic
amino acids the biochemical significance of protein methylation in muscle
tissues is unknown at present (217). Histones are also heavily methylated
and the results suggest that this phenomenon is one of the many factors
which lead to repression of DNA prior to mitosis (217). While methylated
amino acids have been demonstrated in filariae, the proteins are yet to be
identified.
It 1s apparent from the results that filariae are unable to synthesize
methylated bases (Table 10), It is, however, possible that the turnover of the enzyme*
mediating these reactions in filarial worms is so low that a much longer incubation
70
pe ri od s ho ul d have been used. Thi-re a re also d o u b t s a b o u t the p r e s e r ce
of methylated bases in o th e r p a r a ites. A s t u d y of n u c l e i c a ci d s of
P l a s m o d i u m berqhei did not reveal the p r e s e n c e of a n y m e t h y l a t e d bas e s
but it was o b s e r v e d that the e x t r a c t i o n pro cedure was not a p p r o p r i a t e to
r e s ol v e t h e se c o m p o u n d s (183). A t t e m p t s b y A r o n s o n and J a f f e (205) to
demonstrate the p r e s e n c e of 5 - m e t h y l c y t o s i n e in T . e q u i p e r d u m w e r e a ls o
futile. Incorporation of radio act ivi ty from [ C H ^ - ^ C ] m e t h i o n i n e into
the n u c l e i c a c i d f r a c t i o n can p r o b a b l y be r e g a r d e d a s a r t i f a c t u a l . The
p r e s e n c e of 5 - m e t h y l cytosine, however, has b e e n d e m o n s t r a t e d in o t h e r
eukaryotes. It is e s t i m a t e d t h a t b e t w e e n 21 a n d 8 % o f t he c y t o s i n e s of
s p e c i e s f r o m the C o e l e n t e r a , M o ] l u s c a , Ann e l ida , E c h i noderrnata a n d
Chordata a re m e t h y l a t e d (218).
The o b s e r v a t i o n that the a d d i t i o n of p o t a s s i u m c y a n i d e led to an
inc rease in the a c t i v i t y of filarial A d o M e t s y n t h e t a s e is ar. i n d i r e c t
ind ic ati on tha t e x t r a c t s of these p a r a s i t e s e x h i b i t A d o M e t d e c a r b o x y l a s e
p r o p e r t y w h o s e c a t a l y t i c a c t i v i t y p r o d u c e s d e c a r b o x y l a t e d Ado'.'et,t'ne
p r i m a r y role bei ng for the s y n t h e s i s of polyainine ( 2 0 2 , 2 1 9 - 2 2 2 ) .
P r e v i o u s s t u d i e s have s h o w n t hat A d o M e t d e c a r b o x y l a s e a c t i v i t y is
abolished by c a r b o n y l reagents including cyanide, p h en yl h y d r a z i n e end
s e m i c a r b a z i d e (223). Polyainine f o r m a t i o n is c o m m o n in p r o k a r y o t e s and
eukaryotes. Recently b l o o d s t r e a m forms of T r y p a n o s o m a b r u c e i were
reported to p r o d u c e p o l y a m i n e s (224). It is w i d e l y b e l i e v e d that
polyamines have important regulatory roles in m a m m a l i a n cell physiology,
b ut t h e s e a re at p r e s e n t u n c e r t a i n .
71
Other metabolites of AdoMet whose presence tn filariae was not
investigated are briefly considered below because of their physiological
importance. Bacteria, baker's yeast and mammals metabolize AdoMet to
5‘
-methylthioadenosine (MTA) but the microorganisms also produce
amimHp-butyro-lactone (200,144). In Aerobacter aerogenes but not in
yeast, 2-am1no-3-butenoic acid appears to be the initial product in the
formation of butyrolactone (200). Further metabolism of MTA gives
5'-methylthioribose and adenine in bacteria and 5'methylthioribose-l-
phosphate in mammals (144). Adenine is the second product in both systems.
In both prokaryotes and eukaryotes there are speculations that the
methylated thioribose may eventually produce methionine (144). MTA is
considered a potent regulator of enzyme systems and it has been demonstrated
to inhibit proliferating human lymphocytes (144).
The demethylated product of AdoMet, AdoHcy, also goes through multiple
routes of transformation. In bacteria, but possibly not in yeast and
mammals, AdoHcy is cleaved by a nucleosidase to S-ribosylhomocysteine
and adenine (200) the former product undergoing further metabolism to
give homocysteine and ribose (225). There is evidence that the same
bacterial nucleosidase catalyzes the formation of 5*-methylthioribose
from AdoMet. In mamnalian systems, however, the action of L-amino acid
oxtdase on AdoHcy results in the formation of S-adenosy1'^-thio-«t-
ketobutyrate (200). Non-specific adenosine deaminase of Aspergillus oryzae
also deamlnates AdoHcy as well as MTA and in the case of the former
to S-1nosylhomocysteine (200). Although none of these transformed products
of AdoHcy were Investigated in filariae, it is possible that these
parasites may have such mechanisms owing to the widespread nature of

these products of AdoMet.
72
Py far the most c o mmon p at hw ' / o f A d o H c y m e t a b o l i s m in e u k a r y o t e s
is by w ay o f A d oH c y h y dr ol a se (22 ) w h i c h has been i de nt i fi e d in f i l a r i ae
(Tables 2 and 3 ). A do Hc y h y dr o la s e has been e x t e n s i v e l y st ud ie d be ca u se
it m a y be crucial in the r eg ul at i o n of tissue levels of A d o H c y ( 2 2 6
228). The e n zy me has been found in all rat tissues e x am in e d, with
e s p ec i al l y high levels in the liver and p an c r e as (144,186). Mammalian
A doHcy hyd ro l as e app ea r s to have an optimal pH around 6.5 (144).
A p p a re n tl y the same e nz y m e c a t a l y z e s both the forward and rev er se
reactions (186,229). The c o n d e n s i n g e n z y m e seems to e x h i b i t an a b s o l u t e
requirement for h o m o c y s t e i n e w h e r e a s some structural m o d i f i c a t i o n s of
the n uc le os id e can be t ol erated (229,230). Calf liver A d o H c y h y dr o l a s e
was r ece nt ly p ur if ie d to h o m o g e n e i t y (231). Molecular weight determination
of the native e nz y me by g r a d i e n t gel e le c t r o p h o r e s i s in the pr e se n c e of
sodium d o d e c y l s u l p h a t e has s u gg e st e d that A d oH c y h y d r o l a s e m i q ' t be a
tetrai'icr.
It is a p p a r e n t f ro m s p e c t r o s c o p i c stu d i es that calf 1 i \ l r A d o H c y
hy dr ol a s e c on t ai ns a ti gh t ly bound N A D + (231,232) p r o s t h e t i c i-roup; this
NAD*" has been impli ca t e d in the m e c h a n i s m of a c ti o n of the enzyme.
Addition of a d en o s i n e to A d o H c y h y d ro l as e c au s ed an i nc r ea s e in the
a bsorbance at 327 nm w h i c h w as a t t r i b u te d to NADH forma t io n.
Al th o u gh the e q u i l i b r i u m of the r ea c ti on f a vou rs the f u r c a t i o n of
A do H cy (229), the e n z y m at i c removal of h o m o c y s t e i n e and a d e n o s i n e (end
product i nhibitors of the h yd ro la s e ) (200,229) s ho u ld e ns ur e a u n i d i r e c t i o n a l
t ra ns fo r m at io n of m e t h i o n i n e to c ysteine. F il a ri a e c an convert; ade.iosins
t^ p urine nu cl e ot id e by w a y of a d e n o s i n e k i nase or a f t e r its dâ. ;iiu tion
to form inosine and e v e n t u a l l y h y p o xa n t h i ne , by w ay of h y p o x a n t h i n e : ^ u a n i n e

73
phosphorlbosyltransferase (267). The properties of these auxiliary
enzymes have also been Investigated in mammals and microbial systems (233,234).
Homocysteine is utilized in the following reactions:
a. Synthesis of methionine by the vitamin B 12-independent and
dependent pathways (156,160,179,187,200).
b. AdoMet-dependent methylation of homocysteine to methionine (200).
c. Condensation with serine to form cystathionine a reaction irreversible
in mammals but reversible in higher fungi e.g. Neurospora crassa (200,
235,236).
The absence of the first reaction in filariae suggests that filarial
homocysteine can participate in only reactions (b) and (c). Attempts to
show the formation of homocysteine from cysteine (an irreversible
pathway, in at least two species of bacteria (163))met with no success
in the filariae. In mammals and microbes the kinetic constants of the
compone.nts of methionine synthesis suggest that low levels of tissue
homocysteine should favour the activity of N^-methylTHF:homocysteine S-
methyl transferase (144). This presupposes that AdoMet .‘

homocysteine
methyl transferase will be at a kinetic advantage owing to its high
affinity for homocysteine. On the other hand elevation of homocysteine level
would consnit this thiol compound to transsulphuration just like the situation
1n mammalian systems. Of course this depends on whether the filarial
synthase shows a similar kinetic constant; at the time of writing,
insufficient quantities of this filarial enzyme were available to permit
such kinetic analyses.
(d ) AdoMet:Homocysteine S-Meth.yltransferase
Methionine formation by the AdoMet-mediated direct remethylation
of homocysteine has been observed In several biological systems.

74
Law et al. (237) have d e m o n s t r a t e d that A d o M e t :h o m o c y s t e i n e niethy 1 -
tr a ns fe ra s e is present in c e ll - f r ^ e e x t r a c ts from 1-hr Mus ca d o m es t i c a
embryos. A c t i v it y of the t r a n s m e t h y l a s e has also been d e t e c t e d in
Candida u ti li s (230), a n u m b e r o f str ai ns of A . a e r o q e n e s (206),
S.c e r e v i s i a e , E.col i s tr ai n K-12 and m a m m al s (1 30 ,2 3 9, 24 0) and n ow in
filariae.
The level o f the filarial methyl t r a n s fe ra s e is c o m p a r a b l e to that
of the c o r r e s p o n d i n g e n z y m e in m a m m a l i a n liver, w hi c h is the m a j o r s it e o f
m e th i on in e m e t a b o l i s m (144). The filarial t r a n s m e t h y l a s e was p a r t i a l l y
purified w it h a p u r i f i c a t i o n f actor of 5 and a y i e l d o f 9 4%
(Table 4). The p ro pe r t i e s of the filarial e nzyme are s i m i l a r to the
isofunctional m icrobial t r an s m e t h y l a s e with o nl y m i n o r v a r i a ti on s. The
microbial e nz ym e has been shown to be s pe c if i c for the methyl do nor, S-
a d e n o s y l - L - m e t h i o n i n e (241). The d e x t r o - r o t a t o r y methyl sulpr.onium
compound and o t h e r methyl d onors w hi c h are a ct iv e in the thetin-homocystei'.
m et h yl p h e r a s e s y s t e m of liv er w e r e i n ef fe ct i v e (175,176). A l t h o u g h it
was not d e m o n s t ra t ed in the filarial system, it is known in p ro k a r y o t e s
and other e uk ar yo t e s that S - m e t h y l - L - m e t h i o n i ne can s u b s t i t u t e for
AdoMet. Mor eo ve r , the same m e t h y l a s e a p p ea r s to act on both methyl
do nors as d e p i c te d by the c o ns t a n t ratio of a c t i v i t y d u ri n g f r a c t i o n a t i o n
of the e nz ym e from S . c e r e v i s i a e (240). Since A d o M e t is the o n l y s u bs t r a t e
which has been ident i f ie d in animal cells, (S-methylmethionine, apparently
occurs w i d e l y in plants (242)), and it has a Km of S u M for the filarial
t r an s m e t h y 1 ase it is s u gg e s t e d that this c om p o u n d m a y be the natural
substrate for this filarial e n z y m e (Table 11). T wo Michael is c o ns t an t s
for ttie baker's y e a s t t r a n s m et h y l a se for A d o M e t have been r e po rt ed :

75
8.6 x 1Q"4m (241) and 2.2 x 10“3M (243). The a p p a r e n t v ar i a t i o n in the
baker y e a s t t ra n sm e th y l a s e Km m a y be a t t r i b u t a b l e to d i f f e r e n t b at c h e s
of y e a s t w h i c h is not an u n c o m m o n p he n o m e n o n in biological studies. The

_o
Km for M . d o m e s tica e m br yo t r a n s m e t h y l a s e is 2 .8 x 10 “°M (237). The
higher a f f i n i t y e x hi b i t e d by the filarial e nz ym e is c o m p a r a b l e to that
shown by m a m m a l i a n A d o M e t - u t i l i z i n g m e t h y l a s e s (144).
In c ont ra st w i t h the s t e r e o s p e c i f i c i t y of the methyl donor AdoMet:homo
cyste in e methyl t r a n s f er a se is not s t e r e o s p e c i f i c for the thiol acc ep to r .
Both s t er e o is om er s had d e m o n s t r a b l e a c t i v i t y for the b a ke r' s y e a s t
e nzyme (241). But e xt r ac t s of A . a e r o q e n e s could u t i l iz e only the
l ev o ro t at o r y h o m o c y s t e i ne (241). Whi l e this could not be i nv es t i g a t ed
in the filarial system, it was shown that d el e ti o n of L - h o m o c y s t e i n e
de c re as ed the a c ti v i t y d r a s t i c a l l y ( Table 6 ). However, Sh ap i ro e t a l .
(241) have s u gg es t ed that h o m o c y s t e i n e r ac e ma se a c t i v i t y in the p ur i fi ed
ye as t e x t ra ct m i g h t be r e s p o n s i b l e for the action on D - h o m o c y s t e i n e .
Likewise a la c to na se a c t i v i t y has been a s sum ed to e x p l ai n the e f f e c t of
the m e t h y l ase on h o m oc y s t e i n e t h i o l a c t o n e (241). The Km of h o m o c y s t e i n e
for the t r an s me t h y l as e s f ro m S . c e r e v i s i a e and f i la r ia e ere c o m p a ra b le ,
being 2.3 m M (243) and 0.9 m M (Table 9) r e sp ec t iv el y. A Km of 0 .3 2 r.M
has also been reported for the y e a s t m e t h y l a s e by the same g r o u p of
i nvestigators (2 4 1 ).
In addit i o n to f u n c t i o n i n g as a methyl a c ce p t o r , the p r e v a i l i n g
notion is that h o m oc y st e i n e may a ls o be a c t in g as a r ed uc i ng agent.
Shapiro et, a_l_. (241,243) o b s e r v e d that a 15 m i n p r e i n c u b a t i o n of
S .c e re v i si a e e x t ra c t w it h h o m o c y s t e i n e was n e c e s s a r y in o r d e r to ob tain
l in e ar i t y with time. In spite of this p r e i n c u b a t i o n , t h e rate of r e ac t i o n

76
as a f un c t i on of h o m o c y s t e i n e cor e n t r a t i o n w as not p r o p o r t i o n a l .
S u b s e q u e n t ex periments with various reducing agents confirmed the
r educi ng propertie s of homo cystei ne . Mercaptoethanol w h i c h was the m o s t
e f f e c t i v e a p p e a r e d to r e d u c e the y e a s t e n z ym e ; c y s te i n e , t h i o d iglycol
and 2 ,3 - d i m e r c a p t o p r o p a n o l also produced marked i n c re as e in the rate of
r e a c ti on . In the c as e o f the filarial t r a n s m e t h y l a s e it was not i c ed in
the c o u r s e o f par t ia l purification that the m e r c a p t o e t h a n o l in the
e x t r a c t i o n b u f f e r w as r e s p o n s i b l e for the o b s e r v e d thermal activation
f o l l o w i n g p r e i n c u b a t i o n of the e n z y m e at 50°C for 5 r;iin. It was c o n s i d e r e d
that m e r c a p t o e t h a n o l w as e i t h e r p r o t e c t i n g the filarial e n z y n e a g a i ns t
h e a t d e n a t u r a t i o n or a c t i n g as a c t i v a t o r or both. This p r o m p t e d a study
of the e f f e c t of thiol c o m p o u n d s on the a c t i v i t y of this enzyme. Among a variety
i n v e s t i g a t e d , c y s t e i n e w a s a s i g n i f i c a n t l y b e t t e r a c t i v a t o r of the e n z y m e
than e i t h e r t d i t h i o t h r e i t o l , r e d u ce d g l u t a t h i o n i n e or 2 - m e r c a p t o e t h a n o l -
4
T he i n c l u s i o n o f r e d u c e d g l u t a t h i o n e in the i n cu b a t i o n m i x t u r e for
measuring the m a m m a l i a n methyl t r a n s f e r a s e a c t i v i t y (130) s u g g e s t s that
the o t h e r m e t h y l t r a n s f e r a s e s m a y a l s o r e q u i r e thiols for the e x pr e s s i o n
of t he i r optimal activity.
In all t he s e cas es it did n ot a p p e a r t ha t the r e d u c i n g a ge nt s were
a l s o r e p l a c i n g h o m o c y s t e i n e as m e thyl donor. Ariammalian thiolnethyl-
transferase that catalyses the m e t h y l a t i o n of a v a r i e t y of r.ci-physlological
sulphydryl c o m p o u n d s s uc h as m e r c a p t o e t h a n o l has been r ep o rt ed (24 -
246). i n c o ntrast with the y e a s t a nd filar ia l t t a n s m e t h y l a s e s w h i c h are
cytosolic, the a b o v e m a m m a l i a n m ethyl t r a n s f e r a s e is m i c r os o ma l and
i n d e p e n d e n t of d i v a l e n t metal ions for the e x p r e s s i o n o f its c at a l y t i c
activity. T h e i n v o l v e m e n t of met al ions in the c a t a l y s i s of Adul-iet:-
h o m o c y s t e i n e m et h y l t r a n s f e r a s e is v a ri ab l e . The bacterial and y e a s l

77
t ra n sm e th y l a s es were a c t i v at ed by Z n++ and C d + + ; but M n + + , N i++ and C o ++
were wit ho ut e ffect (241). In a d d i t i o n to s t i m u l a t i o n by t ra ns i t i on
d iv a l e n t metal ions, a l ka l i n e ear th metal ions, M g M and C a + + , also
a c ti va te d the filarial t r a n s m e t h y l a s e (Table 7). by contrast the
c o r r es p on d i ng m a m m a l i a n e nz ym e does not a ppe ar to r eq ui r e me ions for
activity, since n e i t h e r EDTA nor 1 ,1O - o r t h o - p h e n a n t h r o l i n e a f f e c t e d its
a ct iv it y (247); both c h el at o r s , h ow ev e r , in hi bi te d the filarial and
microbial t r a n s m e t h y l a s e s ( Table 8 and 247). Even t h ough p a r t i c i p a t i o n
of divalent metal ions in the m e t h y l a s e a c t i v i t y o f f il a ri a e and m i c r o b e s
has been d e m on s tr a t e d , in none of the s e enz y me s has an a b s ol u t e m e t a l l i c
re qu ir e m en t been shown. This seems to c o n f i r m the o b s e r v a t i o n of
M al m st r o m and R o se n b er g (248) that there is no a b s ol u t e s p e c i f i c i t y in
metal ion a c t i v a t i o n of e nz ym e s ; since in all cases i n ve s ti g a te d more
than one metal ion has p r on ou n c e d e f fe c t on the c a ta l y t i c a c t i v i t y of
the protein. However, there are u s u a l l y d i s t i n c t d i f f e r e n c e s in the
ef fi c ie n cy with wh ic h d i f f e r e n t m e t a l l i c ions act as a c ti v a t o r s for
given e nzyme as has been shown for these tr an sm et h yl a s e s .
T he o p t i mu m pH e x h i b i t e d by filarial t r a n s m e t h y l a s e (Fir. 3) is
similar to that of S . c e r e v i s i a e (240); in both cases a ro u n d pH 3.0.
However, the rate of the e nz y m e r ea c t io n has u s u a l l y been m e a s u r e d at pH
values lower than the o p t i m u m so as to a vo i d the chemical decomposition
of AdoMet whi ch takes place in a l k a l in e media (195-198).
The o p t i m u m t e mp er a tu re of filarial Ado?1et:hoiiiocys tei ne methyl -
tra ns fe ra se of 50°C (Fig. 5) a p p e a rs to be u n u s u a l l y high; this m ay be
related to the prese n c e of m e r c a p t o e t h a n o l in the r e a c t i o n i.:ixture as
pointed out p r e v io u s l y in this di sc u s s i on . Stu di es on the e f f e c t of

78
temperature on the analogous enzyme in jack bean meal revealed that
the optimum activity occurred at 60°C (242).
With the exception of glycine methyl transferase which is weakly
inhibited by AdoHcy, all Ado Met-uti1izing transmethylases are potently
inhibited by the thiol ether product (144,249). The effect of AdoHcy on
AdoMetrhomocysteine methyl transferase is uncertain. The microbial
methylase was reported to be inhibited by AdoHcy (241). However, Ferro
and Spence (250) observed that the level of the enzyme in cells of
S.cerevisiae grown in 0.1 mM AdoHcy was unaffected. It could be that
the effective concentration of AdoHcy was not achieved because of its
metabolism in the cells or due to the lack of a transport mechanism of
AdoHcy in the yeast. The filarial and M.domestica transmethylases were
refractory to inhibition (see result section, 237). Although no information
exists on the intracellular levels of AdoMet and AdoHcy in filariae, the
value of 0.045-0.060 umol/g wet weight in mammalian liver (237) suggests
that in v i v o , the filarial transmethylase still would be uninhibited. On
the contrary AdoMet: homocysteine methyl transferase from filariae was
apparently susceptible to inhibition by methionine. It was observed
during studies on the relationship between enzyme concentration and rate
of reaction that a negative slope resulted if the level of AdoMet was
2 mM, It was lucidly demonstrated in S.cerevisiae that there was 50%

79
inhibition of the rate of reacti i w it h as low as 0.4 umole of added
m e t h i o n i n e (241). The rate of incuhionine f o rm a ti o n was l in e ar only if
its c o n c en t r at i on was less than 0.5 umole/ml of r eac ti on m i x t u r e (240).
A lt h ou gh a long p eriod has e l a ps e d since the i n t r o d u c t i o n of DEC
and sur am in in the c h e m o t h e r a p y of f il ariasis, e x a c t l y h ow they e xe r t
their antif il ar i al a ction is still uncertain. The s trong s e n s i t i v i t y of
some f o l a t e -r e l at e d e n z y m e s to the i nh ib i to r y a c tion of these drugs has been
observed. DEC has been d e m o n s t r a t e d to s t r o n g l y i n h ib i t filarial
serine hydroxyinethyltransferases, C H ^ TH F d e h y d r o g e n a s e s , N A D P - i n d e p e n d e n t
10-f or my lT H F d e h y d r o g e n a s e as well as B.pahangi C H g T H F r e du c t a s e (117)
whereas it had marginal or no a c t i v i t y a g a i n s t the c o r r e s p o n d i n g m o s q u i t o
enzymes (122,125). Jaffe and a ss o c i a t e s (57) also found that filarial
di ny d ro f ol a t e red uc ta s e and N A D P - d e p e n d e n t 1 0 - f o r m y l T H F d e h y d r o g e n a s e
were sen si t iv e to inhibi ti on by s ur a mi n; how ev er the i sofunctional
m a mm al ia n and m o s q u i t o d e h y d r o g e n a s e s N A D P - d e p e n d e n t (58) and Mosquito
CH^THF reductase, 1 0 - f o r m y l T H F s y n t h e t a s e (122,123), and thymidylate
synt he ta se (124) w e r e also s t r o n g l y inhibited by suramin. The a c t i v i t i e s
of trypanosomal a - g l y c e r o p h o s p h a t e oxida se , d i h y d r o f o l a t e r ed uc t as e ,
t hy m id yl a t e s y nt he t as e and d i h y d r o o r o t a t e h y d r o x y l a s e w e r e also blocked
by suramin (251,252). In spite of the i n t e r fe r en c e w it h f o la te m e t a b o l i s m
and hence also f o l at e - d e p e n d e n t r ea c ti o n s in filar i ae c aused by these;
f il a ricides these eff ec ts may not r e pr e s e n t their only n od e of action.
Suramin has been d e mo ns t r a t e d to be a p otent i n h ib i t o r of a variety of
other en zy m es including those invol v ed in c a r b o h y d r a t e m e t a b o l i s m and
DMA r e p li ca ti on and t ra n sc ri pt i on (19,253). As the p la s m a concentration
of these f il ar icides is lowe r than the level u t i l iz e d in this stud y

(19,20) it seems u nl i k e l y that in vivo s ur am i n and DEC m i g h t be e x e r t i n g
their anti filarial action, e ve n in part, by in hi b it in g A d o M e t : h o m o c y s t e i n e
methyl t r a n s f e r a s e .
As e a rli er indica ted,a l t ho ug h p r o k a r y o t i c and e u k a r y o t i c cel ls were
found able to form m e t h i o n i n e by the A d o M e t - m e d i a t e d d i r e c t r e m e t h y l ati o n
of homocys t e i ne , the p hy si o l og ic al s i g n i f i c a n c e of this is obscure. It
has been sug ge s t ed that the high t u r n o v e r in the m e t h i o n i n e pool o f the
cell wou l d m ak e A d o M e t : h o m o c y s t e i n e methyl t r a n s fe ra s e a f ai l -s a f e
m e c h a n i s m for the sy n th es is of m e t h i o n i n e should the level of this amino
acid d ec l i n e bel ow a critical poi nt (130,200). It is t h o u gh t that this
will e n sure the f u n c t i o n a l i t y of A d o M e t s y n t h e ta se , w hi c h has a high Km
for me th i o n i ne , the tissue level of wh ic h is low. It has been d e m o n s t r a t e d
that w he n S . c e r e v i s i a e w er e grown u n d er c o n d i t i o n s w he r e the i n t r a c e l l u l a r
c o n c en tr a ti on s of A doMet were e le v a t e d there were r e p r es si o n of three
m e t h i o ni n e metabolic enzymes: h o m o c y s t e i n e synthetase, ATP-sulphurylas
and A d oM e t s y n t he t a se with a c o n c o m i t a n t i nduction of A d o M e t : h o m o c y s t e i n e
methyl tr a n sf er as e (250). It was t he refore s u gg es t ed that the e nz ym e
mig ht regulate A doM et and m e t h i o n i n e b al a n c e and s y n t he si s. It was
further c o ns i d e r e d that this i n d u c t i o n - r e p r e s s i o n p h e n o m e n o n would
p revent the super f l uo us , t hough i n e v i t a b l e - c y c l i z a t i o n of m e t h i o n i n e
which wou ld occ u r were A d o M e t s y n th e ta s e and A d o M e t : h o m o c y s t e i n e m e t h y l -
tr an sf e r as e to op er at e s i mu lt a ne ou s ly .
Law and c o l l e a g u e s (237) have p r o p o s ed that A d o M e t i h o m o c y s t e i n e
methyl tra ns f er as e may be f u n c t i o ni n g to r eg u la t e the a c ti v i t y of the
tRNA methyl t r a n s f e r a s e s . T he y n o t i ce d an inverse r e l a t i o n s h i p bet we en
the activi ti es of these t r a n s m e t h y l a s e s d uring e m b r y o g e n e s i s of M . d o m e s t i c a

C254); the activity of AdoMet .-homocysteine methyl transferase declined
with progression of embryonic development. A similar observation was
made by Kuchino et al_. (255) in hepatoma cells in which elevated levels
of tRNA methyl transferase activity were noticed with a total absence of
Ad oMet:homocysteine methyl transferase. Inverse relationships have also
been established between glycine-N-methyltransferases and tRNA methyl-
transferases in neoplasms, fetal rabbits and aging rodents (249,254,256).
AdoMet-Mediated Methyl transferases as Potential Targets in the Chemotherapy
of Parasites: As indicated elsewhere in this text, transmethylation
reactions utilizing AdoMet are sensitive to inhibition by AdoHcy.
It has been shown that the sensitivity of these methylases is greatly influenced
by changes in AdoMet/AdoHcy ratio (144). The activity of AdoHcy hydrolase
is greater than that of AdoMet synthetase in all cells examined so far and
the concentration of AdoMet is usually 3 to 4 times larger than that of AdoHcy.
On the basis of these findings emphases have been directed to designing
analogues that would interfere with AdoMet/AdoHcy ratio ultimately leading
to the control of the utilization of AdoMet.
One approach has been to alter the activity of AdoMet synthetase.
Inhibition of this enzyme would lower the concentration of AdoMet thereby
inhibiting all AdoMet-dependent reactions. Of the several analogues of
methionine that were studied, 1 -aminocyclopentane- 1 -carboxylic acid
(cycloleucine) and 2-amino-4-hexynoic acid proved potent (203,257,258),
AdoMet analogues have also been evaluated for their activity
against AdoMet-dependent methyl transferases. Sinefungin and A9145C
isolated from Streptomyces griseolus were the outstanding members of
this group of inhibitors (146),

82
But the approach which has attracted more considerable attention
is reduction of the activity of AdoHcy hydrolase which will result in an
increase in the concentration of AdoHcy. An analogue which is a powerful
inhibitor of AdoHcy hydrolase is 3-deazaadenosine and its administration
has been shown to produce pronounced biological effects such as decrease
in creatine and phosphatidylcholine in rat liver (259), antiviral activity
against Rous sarcoma virus in chick embryo fibroblasts and Gross murine
leukemia virus in mouse embryo cells (260). 3-Deazaadenosine has also
been shown to produce antimalarial activity against Plasmodium falciparum
in culture (259).
Recent findings indicate that AdoMet-dependent methylations might
regulate important physiological activities whose study in filariae
and related parasites may likely furnish answers to some of the baffling
biochemical and physiological peculiarities which characterize the
parasitic adaptation. Biological methylation appears to be important in
parasites in the area of phospholipid synthesis and protein modification.
Phospholipids are a major component of biomembranes and provide the
fluid matrix for movement of other membrane components (144). The
cuticle of filariae, as of other parasites has high level of lipid of
which phospholipids are among the dominant ones (113)i Ascaris lumbricoides
cuticle, for instance, is composed of 38% phospholipids (107). The
heavy demand of filariae for phospholipids is indicated by the absence
of betainerhomocysteine methyl transferase (Table 5) which has been
suggested by Finkelstein elt.a]_. ,(261) to metabolize excess homocysteine
and betaine. It has been observed that parasites readily alter their
outer coat to make immunological recognition less possible. Extensive

83
a n t i g en ic va ri at i o n has been noti: ?d in t r y p a no s o m e s ; d i f f e r e n t c lo ne s
of T.brucei have been found to coi.tain d i v e r s e g l y c o p r o t e i n s (262). In
s chistos om es , e le ct r o n m i c r o s c o p y s u g g e s t s that the v i ti at i on of host
r es ponse de pe nd s on c on ti n u o u s m e m b r a n e a c t i v it y at the s c h i s t os o m e
integument. It is l ikely that these me mb ra na l a c t i vi ti e s are a s so c i a t e d
w it h a rapid t ur nover of the p ar a si t e s p h o s p ho l ip i d s . In a d d i t i o n there
should be a clo s e p a r a l l e l i s m between the se c u t i c u l a r c h a n ge s and
p h o s p h a t i d y l c h o l in e - l y s o p h o s p h a t i d y l c h o l i n e i n t er c o n v e r s i o n or p h o s p h o l ipases
A1 and A2 a c t i v it i es . L y s o p h o s p h a t i d y l c h o l i n e r el e a s ed by the a c ti o n of
these lipases is lately being regar de d as a natural f u s i g en f using the
lipid b il a ye rs together. It is p oss ib le then that p e r t u r b a t i o n of
p arasites A d o M e t / A d o H c y ratio c o u p l e d w it h i n h i bi t i on of p h o s p h o l i p a s e s
A1 and A2 m i g h t help i n te rr u pt the p e r i od i c t r a n s f o r m a t i o n of the
surface coat.
A doMet is further involved in the r u d i m e n t a r y ne rv ou s s y s t e m of
parasites. A c e t y l c h o l i n e is an i n h ib i to r y n e u r o t r a n s m i t t e r in S .nansoni
(111). Its prese nc e in f il a ri ae has been inferred from the d e t e c t i o n of
c h o l i nesterase a c ti v i t y in D . v i t e a e (44). S e ro to ni n acts as a s t i m u l a t o r y
tr an sm i t te r in ad di t i o n to its r e g u l a t o r y role in the c a r b o h y d r a t e
m e t a b o l i s m of t rem at od e s (111). M o re o v e r , s p h i n g o m y e l i n w h i c h is a
c o n s ti tu e n t o f tiie nervous t is s ue has been r ep o r t e d to be p r e s e n t in
some p ar asites including filariae (102, F ig .12). T he s e s ub s ta nc e s ,
however, are p artly e it he r p rod uc ed or d e g r a de d thr ou gh m e t i y l a t i o n , for
instance d e p o l a r i z at i on and g lu co s e d e pl et i on m a y o c c u r in f l at w o r m s if
the A d o M e t - me d ia t e d c a t a b o l i s m of s er o to n i n is blocked.
84
Like phospho 1 i pi d s , pro te in :■e th yl a tion is also of vital i m po r t a n c e
to the cell, a lt h ou gh little is k'.own about this p r oc e ss in p ar as it es .
M o d i f i c a t i o n of protein by m e t h y l a t i o n is a s s o c i at e d w i th m a n y d iv er se
p henomena such as bacterial and l e u co c yt e c he m ot a x i s , and pept id e
hor mo ne d e g r a da t i on and n e u r o s e c r e t i o n (144).
Tfie e nz y m a t i c methyl e s t e r i f i c a t i o n of the m e m b r a n e p o l y p e p t i d e is
an area wor th in ve st igating. Such a r ea ct i on will r e s u l t in c h a r g e / p o t e n t i a l
a l te r at io n of the m e m b r a n e and m a y inf lu en c e c h a r g e - r e l a t e d m e m b r a n e
function, shape and s tr u ct u r e of the cuticle. This w o u l d a ff e ct the
t r a n s cu t ic u l ar u ptake of n u t r i e n t w hi c h Chen and H o we l ls (115) have
su gg es t e d m i g h t be r e l e va nt in filariae. Inte re st i ng ly , the o nl y m a j o r
m et h y la t ed p o l y pe p ti d e in r abbit e r y t h r o c y t e m e mb r a n e s has a ls o been
impli ca te d as a c o m p o n e n t of the g l uc os e tra n s po rt s y s t e m of the e r y t h r o c y t e
(144) w h i c h like filariae and o t h e r h e l m i n t h p a r as it e s d e p e n d s p r i n c i p a l l y
on p h os ph o ry l a t i ve g l y c ol y si s for energy. E q u al l y i m p or ta nt in filarial
worms is m e t h y l a t i o n of pro te i n lysyl and arginyl r e sidues. Methylation
of lysyl g roups is m e d i a t e d by p r ot e in m e t h y l a s e I l l w h o s e a c t i v i t y is e l e v a t e d
in he av il y p ro l i f e r a ti n g cells (145). As female niacrofi l a riae have a
high r ep r o d u c ti v e c a p a c i t y they should have a high a c t i v i t y of this
enzyme. M i c r o f i l a r i a e and o t h e r d e ve lo pm en t a l stages of p a r a s i t e s
should also possess c o n s i d e r a b l e amounts of the enzyme. S e le c t i v e
inhibition of this e nz y m e mig ht be detrimental to the filarial worms.
C he m o t n ct i c responses of o r g a n i s m s to harmful and b eneficial
environ m e n ta l subst a n c es have been linked to the c o m p l e t i o n of the
life cycle of helmi nt h p a r as i te s e s p e c i a l l y those w it h free living
s tages (263). Ami no acids have been impli ca te d as the stimuli a t t r a c t i n g
m i r a c i d i a of schist os om e s to their m o l Tuscan vcctors (259). This lias

85
led to the sugges ti on of the incl '.ion of amin o acid a n al o g ue s into
l ar vi ci d e s to dec o y the m ir a c i d i a . F ur th er mo r e, pro l i ne and g l ut a m a t e
have been i dentified as chemical a t t r a c t a n t s for B i o m p h a l a r i a g l a b r a t a ,
the snail host of S.mansoni (263). It is thus p os si b le to u se these
amino acids as baits to trap the snails. Ch e mo ta xi s , however, is an
A d o M e t - d e p e n d e n t t r a n s m e t h y l a t i o n pro ce ss (144). It has been d e m o n s t r a t e d
in both p r ok ar y o te s and e u k a r y o t e s that in the p re se nc e of an a t t r a c t a n t
there is an incre as e in the m e t h y l a t i o n of s o -c al l e d M e t h y l - a c c e p t i n g
C hemotaxis P r o t ei n (MCP) lo ca te d in the c y t o p l a s m i c m e m b r a n e wh er e as the
c on verse takes pl a ce with the r e p e l l e n t (144). T he s e r e a ct i o ns pre ce de
the m o ve me n t of the o r g a n i s m in response to the st imulus. The o b s e r v a t i o n
that m e t h y l at i on d e f i c i e n t m u t a n t str ai ns or cells d e p le t e d of A d o Me t
are d e fe ct i ve in c h e m ot a x is (144) m ea n s that i n t e r f e r i ng w it h the
parasitic h e lmi nt hs m e t h y l a t i o n a p p a r a tu s es m i g h t y i e l d e f f e c t i v e
results even though the e nv i r o n m en t al stimuli n ay be available.
The s t a b i li t y of m R N A d e p e n d s on its m e t h y l a t e d cap stru ct u re ,

7 51 51
m G ppp N, at the 5 '- t e r m in u s (144). C ap pe d mRN As f a c i l i t at e i n i t ia ti o n
of protein syn th es i s by p r om o t i n g m R N A b i n d i n g to ribosor.es. Ado Me t -
u ti lizing tRNA m e t h y l a s e s are among t RN A m o d i f y i n g e n z y me s that c o n f e r
species d i ff er e n ce s to the h i gh ly c o n s e r v a t i v e s e c o n d a r y s t r u c t u r e of
tRNAs (145).
It seems i nc on c e i v a b le that w i t h o nl y f ou r m a j o r n u c l e o t i d e n i t r o g e n o u s
bases g iving a tri pl et code of 64 c od on s there s hould e vo lv e wide p h y s i o lo g ic a l
and mo r p ho lo gi ca l variations in nature. D NA m e t h y l a t i o n w h i c h o ccurs
prior to and i mm ediately a f t e r the S - p h a s e is thought to e x p la i n this
paradox. It has been su gg es te d that it w o ul d a ls o p r ote ct the O N A
ag ai n st integration w i t h a fore ig n D MA and the a c ti on of D NA as es ; as,

86
for example, bacterial restriction and modification enzymes which are
mechanisms for the recognition and degradation of invading foreign DNA.
Although no radioactivity was detected in the nucleic acid fraction
of D.iimiltls (Table 10) during the six-hour period of incubation in
(CH^-^cJmethionlne, the current notion that modification of nucleic
acid by AdoMet-dependent methyl transferases is conmon in prokaryotes and
eukaryotes (146) makes this area in filariae worth pursuing because if
there exists a-species-specific DNA methylation pattern, which seems
plausible, 1t might be an exploitable site for antiparasitlc chemotherapy.
There is no doubt from the foregoing that AdoMet-mediated methylation
reactions may offer new insights Into the physiology and biochemistry of
filariae and related parasites; but one wonders whether with the versatility
and significance of these reactions, usage of methyl transferase inhibitors
would provide any selectivity. For instance, inhibition of AdoMet
synthetase and AdoHcy hydrolase will result in a general inhibitory
effect on all AdoMet-dependent methyl transferases. Nevertheless, the
situation with dihydrofolate reductase inhibitors 1s a perfect analogy.
Although dihydrofolate reductase is ubiquitous, a selective effect on
rapidly proliferating cells by these antifolates forms the basis of
their action (264). Therefore the knowledge gained with the use of
folic acid analogues can be extended to AdoMet-dependent methylation
reactions. For example, as the ability of some parasites to evade the
host irrroune systems implies a high membrane turnover, and with phospholipids
being one of the major constituents of membranes, it is envisaged
that parasites will be more prone to the adverse effect of
phospholipid methylation inhibitors. As with dihydrofolate reductases
the following factors should be investigated, namely: (1) relative rates
and transport mechanisms of metfiylases inhibitors between host and.

87
parasite, (2) degree of binding and (3) relative rates of metabolism of
inhibitors in host and parasite, and (4) existence of isoenzymes with
different binding characteristics.
Since methylation reactions in living systems are numerous,the prospect
of achieving selectivity on the surface appears remote as a whole array of
Important reactions may be blocked. It has, however, been reported that in
humans about 89% of the net flow of methionine through AdoMet 1s involved
in the formation of creatine (265,266); guanidoacetate methyl transferase
which catalyzes this reaction has not been detected in any invertebrate (266).
Although this difference is important, it is still not enough to afford
selectivity. Being an AdoMet-dependent enzyme, guanidoacetate methyl transferase
should be affected by AdoMet-mediated methyl transferase inhibitors directed
against enzymes of the infective agent. Thus exploitation of the absence of
guanidoacetate methyl transferase in parasites seems impossible at this stage.
However, kinetic differences between parasite and definitive host AdoMet-
dependent methyl transferases could buttress the above basic variation
between the two groups of organisms. As such kinetic parameters are
non-existent, it 1s important that AdoMet-dependent methyl transferases 1n
parasites and thefr primary hosts be actively investigated. These studies
could yield some of the valuable kinetic differences that could be an
asset chemotherapeutlcally. Hence, just as it has been achieved with
dihydrofolic acid reductases, there is every chance that diligent study of
biological methylation reactions and other aspects of methionine metabolism
In parasites should provide exploitable leads to the synthesis of selective
inhibitors.
In suimary, adult filariae apparently cannot biosynthesise methionine,
they, however, retain the catabolic capacity.

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0692 1078 5416 6

METHIONINE METABOLISM IN FILARIAL WORMS

Robert Asare Acquaah

Date: September, 1980

Dr. K.K. Oduro, Internal Supervisor

&r. J.J. Jaffe, External Supervisor

Thesis submitted to the Department of

The experimental work for this thesis was

conducted at the University of Vermont, U.S.A.,

under the direct supervision of Or. J.J. Jaffe

of the Department of Pharmacology.

LIST OF ABBREVIATIONS vii

LITERATURE REVIEW AND INTRODUCTION 3

A. Life Cycle of Filariae 3

C. Socio-Economic Aspects of Filariasis 5

(c) Antimony Compounds 14

(d) Arsenic Compounds 15

(h) Vector Control 17

(i) Sanitation, Urban Environmental

(ii) Chemical Control 18

(iii) Biological Control 19

(iv) Genetic Control 19

2. MATERIALS AND METHODS **

A. Delineation of Methionine Metabolic Pa thway in Filariae 28

(a) N ^- M e t h y l T H F :homocysteine S-Methy lt ra ns fe ra se 30

(b) Betaine:homocysteine S- me th yl tr an sfe ra se 30

(c) ATP:methionine S-Adenosylt ra ns fe ras e 30

(d) S-Adenosylhomocysteine ( A do M et ):homocysteine

(e) S-Adenosylhomocysteine (AdoMet)hydrolase 31

D. Metabolic Fate of AdoMe t in Filariae 33

(a) Extraction of Protein 33

(b) Extraction of Nucleic Acids 34

(c) Extraction of Lipids 34

A. Biosynthesis of Methionine by Filariae 36

B. A bility of Filarial Worms to Metabolize Met hi on in e 37

C. Partial Characterisation of S-Adenosylmethionine:

(a) Int ra ce l1ular Localization of AdoMet:

(b) Dependence of Enzyme A ctivity on Time and

(c) Effect of pH and Ionic Strength on Enzyme

(d) Effect of Temperature on Enzyme Activity. 42

(e) Requirement of Assay. 42

(f) Effect of Divalent Metal Ions and EDTA. 46

(g) Kinetic Studies. 47

(V) Effect of Inhibitors 47

D. Metabolic Fate of AdoMet in Filariae 50

4. DISCUSSION AND CONCLUSION 58

(a) N®- MethylTHF:Homocysteine

S-Methyltransferase and Betaine:

(b) ATP .-Methionine S-Adenosyl transferase. 63

(c) Metabolic Functions of S-Adenosylmethionine. 65

(d) AdoMet:Homocysteine S-Methyltransferase. 73

C. AdoMet-Mediated Methyl transferases as

Potential Targets in the Chemotherapy of

1. Biosynthesis of Methionine by Filariae ^

I. Ability of Filarial Worn^ to Metabolize Methionine

3. Specific Activities of Some Filarial Methionine

4. Partial Purification of Filarial Adc^et iHcwoc'/steine

5. Intracellular Localization of AdoMetsHomocystelne

6. Requirement of Assay <v Filarial Ado^a*ilJonPcysteine

7. Effect of Metal Ions on Filarial AdoMet:Homocysteine

8. Effect of EDTA on Fllflnal AdoMet;Homocysteine

9. Kinetic Parameters cf Filarial AdoMet:Homocysteine