FEMS Microbiology Letters 177 (1999) 39^45

An unusual pattern of invertase activity development in the

thermophilic fungus Thermomyces lanuginosus
Amitabha Chaudhuri 1 , Girish Bharadwaj, Ramesh Maheshwari *
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

Received 3 June 1999 ; received in revised form 7 June 1999 ; accepted 7 June 1999

Abstract

In the thermophilic fungus Thermomyces lanuginosus, invertase displays an unusual pattern of development: the induced
activity begins to diminish even before any substantial quantity of sucrose has been utilized or an appreciable amount of
biomass has been produced. Despite this pattern of invertase activity, neither the growth rate nor the final mycelial yield is
affected adversely. T. lanuginosus invertase is a thiol protein and the enzyme is active when specific sulfhydryl group(s) is in the
reduced state. Measurements of reduced coenzyme and glutathione pools in sucrose-grown mycelia excluded oxidative stress as
the primary reason for the observed decline in invertase activity. Rather, this unusual pattern of invertase is considered to be
due to its localization in the hyphal tips. At the early stage of growth, the number of hyphal tips per unit mass of mycelium is
maximum, whereas at later times their numbers do not increase in proportion to the biomass. As a result invertase activity
shows an apparent inverse relationship with biomass. The enzyme activity disappears when the inducing carbon source is
consumed and growth is completed. 1999 Federation of European Microbiological Societies. Published by Elsevier
Science B.V. All rights reserved.

Keywords : Invertase ; Regulation; Redox status; Hyphal tip; Thermophilic fungus ; Thermomyces lanuginosus

1. Introduction peculiar behavior: when T. lanuginosus was grown in

Our earlier studies revealed that in contrast to
yeasts and mesophilic molds, invertase in the ther-
mophilic fungus Thermomyces lanuginosus was an
induced enzyme, intracellular, highly unstable in
the cell-free extracts [1] but stabilized by thiol com-
pounds [2]. Moreover, the enzyme displayed a very
* Corresponding author. Fax: +91 (80) 334 1814;
E-mail: fungi@biochem.iisc.ernet.in
1
Present address: Department of Molecular and Cellular
Biology, Harvard University, Cambridge, MA 02138, USA.
a liquid medium containing sucrose as the carbon
source, invertase activity was induced rapidly but
the increase in enzyme activity was transient [1].
The enzyme activity began to diminish even before
an appreciable utilization of sugar or any substantial
increase in biomass had occurred. Surprisingly, de-
spite the abrupt decline in invertase activity, neither
the growth rate, nor the nal mycelial yield was af-
fected adversely [3]. By contrast, in the mesophilic
fungi, for e.g. Neurospora crassa [4] and Aspergillus
niger [1], mycelial invertase activity steadily increased
with the age of cultures and was maintained at high
levels even after the carbon source was completely

0378-1097 / 99 / $20.00 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 2 8 6 - 4

FEMSLE 8879 15-7-99

40 A. Chaudhuri et al. / FEMS Microbiology Letters 177 (1999) 39^45
exhausted. In the present investigation we have at-
tempted to explain the unusual pattern of invertase
activity in T. lanuginosus.
2. Materials and methods
2.1. Organism and culture conditions
T. lanuginosus, strain RM-B, was isolated from
horse-dung in our laboratory and has been deposited
in the American Type Culture Collection (ATCC
44008). It was grown in a sucrose-asparagine me-
dium in 150 ml medium in 500 ml Erlenmeyer asks
at 50C with shaking at 240 rpm [3].
2.2. Measurement of biomass and estimation of
sucrose
Samples of culture were removed aseptically at
regular intervals, ltered through a glass ber lter
and washed. Growth was monitored as increase in
mycelial dry weight. Sucrose in culture medium was
estimated using yeast invertase as described previ-
ously [1].
2.3. Assay of enzyme activities
Mycelia were frozen in liquid nitrogen and pow-
dered by grinding with acid-washed quartz in a mor-
tar with pestle. The powder was stirred in 50 mM
Na/K phosphate buer (pH 6.0) containing 1 mM
dithiothreitol (DTT) and 1 mM EDTA for 5^15 min
at 4C. The crude homogenate, or a claried extract
prepared after centrifugation at 12 000Ug for 10
min, was used for the measurement of invertase ac-
tivity by quantitating the reducing sugar liberated
from the hydrolysis of 40 mM sucrose in 50 mM
Na/K phosphate buer (pH 6.5) for 5 min at 50C.
Reducing sugars were estimated by the method of
Somogyi [5]. One unit of invertase activity was de-
ned as the amount of protein that produced 1 Wmol
of glucose per min under the assay conditions.
For trehalase assay, aliquots of the crude homo-
genate were used as the enzyme activity remained
associated with the insoluble matter. Trehalase activ-
ity was measured using 2 mM trehalose in 50 mM
Na acetate buer, pH 5.5. Reaction was done at
FEMSLE 8879 15-7-99
50C for 15 min with intermittent shaking, and the
reducing sugar (glucose) released was estimated by
the Somogyi method. One unit of trehalase activity
was dened as the amount of protein that produced
1 Wmol of glucose per min.
For measurement of G6PDH activity, extracts
were prepared as above in 50 mM Na/K phosphate
buer (pH 7.0) and claried by centrifugation before
use. The reaction mixture in a total volume of 1 ml
contained 12 mM glucose 6-phosphate, 0.1 mM
NADP and 10 mM MgSO4 W7H2 O in 50 mM Na/
K phosphate buer at pH 7.0. The reaction was
initiated by adding dierent volumes of cell extract
and monitoring the increase in A340 due to the for-
mation of NADPH for 5 min at 50C. One unit of
enzyme activity was dened as the amount of protein
that produced 1 Wmol of NADPH per min under the
assay conditions.
2.4. Estimation of total thiols, GSH and GSSG
Mycelia (120^130 mg dry wt) were extracted in
5 ml of 5% sulfosalicylic acid for 10 min in a boiling
water bath. The supernatant obtained after centrifu-
gation was taken for the estimation of total thiols by
reaction with 5,5P-dithio-bis(2-nitrobenzoic acid)
(DTNB). Glutathione (GSH) and glutathione disul-
de (GSSG) were estimated by two methods. The
rst method was based on DTNB-GSSG reductase
recycling assay [6]. The principle of this method is
the oxidation of GSH by DTNB to give GSSG with
stoichiometric formation of thionitrobenzoic acid
(TNB) which is quantitated by measuring absorb-
ance at 412 nm. GSSG was reduced to GSH by
the action of glutathione reductase and NADPH re-
sulting in the formation of more TNB, which was
followed continuously at 412 nm. The amount of
GSH in extracts was determined from a standard
curve where GSH equivalents were plotted against
the rate of change of absorbance at 412 nm. For
the determination of GSSG, GSH in extract was
blocked by reaction with N-ethylmaleimide (NEM).
The unreacted NEM was removed by extractions
with diethyl ether and sample was taken for assay
as before.
The second method was based on the reaction of
thiols with monobromobimane (mBBr) to yield
highly uorescent thioether derivatives which are
 A. Chaudhuri et al. / FEMS Microbiology Letters 177 (1999) 39^45
separated by HPLC and detected by a uorimeter
[7]. For the estimation of GSSG, GSH in extract
was modied by reaction with NEM, followed by
reduction of GSSG with DTT. The resulting GSH
was derivatized by monobromobimane (mBBr) and
samples were analyzed by HPLC.
2.5. Estimation of NADP+ and NADPH in mycelia
The oxidized and the reduced pyridine nucleotides
were estimated according to the method of Klingen-
berg [8]. The NADP in the neutralized extract was
estimated enzymatically by the glucose 6-P dehydro-
genase reaction. The reaction mixture in 50 mM Na/
K phosphate buer (pH 7.0) contained 5 mM glu-
cose 6-phosphate, 5 mM MgSO4 W7H2 O and dierent
volumes of the extract in a total volume of 2.1 ml.
The reaction was started by adding 10 units of
glucose 6-phosphate dehydrogenase (G6PDH) (1
unit = 1 Wmol NADPH formed min31 ) and the in-
crease in A340 due to NADPH was monitored for
20^25 min at 30C.
For the estimation of NADPH, mycelium (280^
300 mg dry wt equivalent) was extracted in 4 ml of
0.5 M KOH in 50% ethanol for 10 min in a boiling
water bath. After centrifugation, the claried extract
was brought to pH 7.8 and NADPH was specically
estimated by the glutathione reductase reaction. The
reaction mixture in 50 mM Na/K phosphate buer
(pH 7.5) contained 0.025 mM GSSG and dierent
volumes of the extract in a total volume of 2 ml. The
reaction was started by adding 0.5 unit glutathione
reductase (1 unit = 1 Wmol NADPH utilized min31 )
and the decrease in A340 due to conversion of
NADPH was monitored for 5^10 min at 30C.
In the second method of NADPH estimation, the
supernatant from alcoholic KOH extraction was ad-
justed to pH 7.8 and the reduced pyridine nucleoti-
des were oxidized using glutamate dehydrogenase.
The extract was deproteinized with 0.2 ml of 3 M
HClO4 and the precipitated protein was removed by
centrifugation. The supernatant was adjusted to pH
7.2 and taken for the estimation of NADP by the
G6PDH reaction described earlier.
2.6. Activity of pentose phosphate pathway
The activity of pentose phosphate pathway was
FEMSLE 8879 15-7-99
41
measured based on the rationale that only the car-
bon-1 of glucose is decarboxylated through this
pathway. Washed mycelia (1 g wet wt) were sus-
pended in 50 ml medium containing 10 mM unla-
belled glucose, 0.1% K2 HPO4 and 0.05%
MgSO4 W7H2 O. One ml of the mycelial suspension
was added to 2 ml of the above medium in Warburg
asks which contained a folded lter paper soaked in
6 M KOH in the center well. After 2 min equilibra-
tion at 50C in a shaker water bath, 14 C-1 glucose
(0.25 WCi) was added to the suspension and the
asks were stoppered. After incubation for 30 min,
the radioactivity due to 14 CO2 absorbed on the lter
paper was measured by scintillation counting.
2.7. Incorporation of 3 H-thymidine in mycelia
T. lanuginosus was grown in a sucrose-asparagine
medium and the mycelia were harvested at 6 h inter-
vals. At each time point similar amount of mycelium
(50 mg wet weight) was suspended in 10 ml of the
spent medium and incubated in a shaker water bath
at 50C. 3 H-thymidine (10 WCi) was added and after
30 min incubation, the mycelium was washed succes-
sively with 5% TCA, ethanol and a mixture of chlor-
oform:ethanol:diethyl ether (1:2:1 v/v) on glass ber
lter. The radioactivity incorporated in mycelium
was determined as described earlier [1].
3. Results
3.1. Distinct patterns of invertase and trehalase
development
Invertase activity in T. lanuginosus grown in a me-
dium containing sucrose as the carbon source, ex-
pressed either as specic activity or total activity,
increased abruptly from undetectable levels to max-
imum level in 6 to 12 h (Fig. 1). Thereafter, the
activity immediately began to fall although nearly
80^85% of sucrose was still available. Signicant in-
crease in biomass occurred during the time when
invertase activity was declining. Despite this fall in
enzyme activity, the rates of growth and utilization
of sucrose were related. As seen from Fig. 1, the time
of half-maximal utilization of sucrose and of half-
maximal growth was the same. The time of complete
42 A. Chaudhuri et al. / FEMS Microbiology Letters 177 (1999) 39^45

the label incorporated per unit mass of mycelia re-

mained virtually constant although the biomass in-
creased up to 36 h. The incorporation of radiola-
belled thymidine did not parallel the increase in
biomass. The time of maximal invertase activity
(Fig. 1) closely approximated the time of DNA syn-
thesis (Table 1) rather than the bulk increase in bio-
mass.

3.3. Reciprocal modulation of invertase activity in

extracts by reduced and oxidized glutathione

Fig. 1. Patterns of development of invertase and trehalase activ-

ity in Thermomyces lanuginosus in relation to biomass and su-
crose utilization. The fungus was grown in a medium containing
2% (w/v) sucrose in shake asks at 50C. Both enzyme activities
were measured in crude mycelial homogenate. Enzyme activity in
total mycelial biomass sampled (20 ml) is referred to as the total
activity.
utilization of sucrose (36^42 h) coincided with the
disappearance of invertase activity. In marked con-
trast, trehalase activity was low as long as carbon
source remained in the medium and it was dere-
pressed after carbon source was exhausted.
3.2. Invertase activity is correlated with the time of
maximal DNA synthesis
In T. lanuginosus invertase is a thiol protein and its
activity in cell-free extracts is modulated reciprocally
by reduced and oxidized glutathione [2]. The redox
status (SH/SS ratio) of the mycelium could, there-
fore, regulate the activity of invertase. To examine
this, an in vitro approach was taken in which the
response of the inactivated enzyme to mixtures of
GSH and GSSG was studied. The activation of in-
vertase depended on the ratio of [GSH]/[GSSG]; the
enzyme activity increasing with the increasing ratio
of SH/SS. Moreover, enzyme activation depended
also on the absolute concentration of glutathione.
At a higher absolute concentration of glutathione,
7.5 mM instead of 2.5 mM, a lower [GSH]/[GSSG]
ratio resulted in higher recovery of activity.

As invertase activity was not correlated with my-
celial dry weight, we determined if it would relate to
growth estimated by DNA synthesis. Maximum in-
corporation of 3 H-thymidine (cpm mg31 dry wt) oc-
curred at approximately 6 h and decreased by 33% in
12 h, by 40% in 18 h and by 70% in 24 h. After 24 h,
3.4. Intracellular redox status
The fall in invertase activity in T. lanuginosus after
its early rise could be because the intracellular envi-
ronment becomes oxidizing with time, i.e. the ratio
GSH/GSSG becomes low, resulting in the oxidation

Table 1
Total thiols, GSH and GSSG in Thermomyces lanuginosus during growth in a medium containing sucrose
Time of growth Total thiols GSH GSSG GSH/GSSG
(h) (Wmole g dry wt31 )
A B A B A B Average
6 6.8 2.0 6.1 1.0 4.8 0.05 0.01 0.06 127 80 104
12 4.8 1.0 3.8 0.5 4.3 0.05 0.01 0.08 72 54 63
18 4.4 1.7 3.6 0.7 2.5 0.07 0.02 0.06 49 41 45
24 4.5 0.3 3.6 0.5 3.6 n.d. 0.08 n.d. 45 45
30 n.d. n.d. 3.5 n.d. 0.06 n.d. 58 58
36 3.7 0.1 3.5 0.5 3.4 0.06 0.02 0.06 59 56 58
A, estimations by DTNB-GSSG reductase recycling assay, in Wmol g31 dry weight.
B, estimations by monobromobimane labelling method, in Wmol g31 dry weight.
n.d., not determined.

FEMSLE 8879 15-7-99

 A. Chaudhuri et al. / FEMS Microbiology Letters 177 (1999) 39^45
of invertase thiols and inactivation of the enzyme.
Therefore, the concentrations of GSH and GSSG
in mycelia were determined. Nearly similar values
were found by both enzymatic and HPLC methods
(Table 1). Throughout, glutathione constituted most
of the total thiol compounds in the cell extracts.
GSSG, which was detected in low amounts, did
not vary signicantly. A high GSH/GSSG ratio
was observed at 6 h when invertase activity was in-
creasing. This ratio was V50 or more during the
growth period.
3.5. GSH levels, NADPH and activity of pentose
phosphate pathway
The cellular level of GSH is maintained by the
glutathione reductase reaction that converts GSSG
into GSH using NADPH as the reductant [9].
Whether the GSH/GSSG ratio is reected in the ra-
tio of NADPH and NADP was determined. The
concentration of NADPH was highest at 6 h and it
declined by 2.5-fold between 6 and 36 h of growth
although the level of NADP did not change signi-
cantly (Table 2). The NADPH/NADP ratio de-
creased 4-fold between these time points.
Since NADPH is generated by the pentose phos-
phate pathway, we determined whether the activity
of this pathway relates to the concentrations of re-
duced NADP coenzyme and of glutathione. The ac-
tivity of pentose phosphate pathway during growth
was estimated by two experimental approaches: by
measuring the decarboxylation of glucose using
14
C-1 glucose and by determining the activity of
G6PDH, a key enzyme of this pathway. As shown
Table 2
Activity of pentose phosphate pathway and the levels of reductive power in Thermomyces lanuginosus during growth in sucrose mediuma
Time of Activity of pentose phosphate pathway
growth (h) as estimated bya
C1 decarboxylation G6PDH
(cpm mg31 dry wt) (units g dry wt31 )
6 1029 26
12 310 20
18 129 17
24 76 15
30 41 14
36 326 17
a
Average values of three determinations from a single experiment.
b
n.d., not determined
FEMSLE 8879 15-7-99
43
in Table 2, both the decarboxylation of glucose as
well as the activity of G6PDH were most active at 6 h
and declined thereafter.
4. Discussion
In fungi, two contrasting developmental patterns
of invertase activity have been observed: (1) The
(constitutive) enzyme activity increases steadily with
time and is highest in mycelium when growth is com-
pleted and the carbon source has been utilized, as in
N. crassa [4]. This pattern was explained on the basis
that a major portion of invertase is distributed in the
intramural space [10] and the increase in enzyme
activity reects the amount of wall material in old
mycelium. Similar pattern was also seen for the in-
tracellular trehalases in N. crassa [11], and in T. la-
nuginosus (present study). (2) The (induced) invertase
activity shows an inverse relationship with the
amount of biomass and disappears upon the exhaus-
tion of the carbon source (sucrose), as in T. lanugi-
nosus (Fig. 1). The present investigation has at-
tempted to understand the distinctive pattern of
invertase in T. lanuginosus.
Because the activity of T. lanuginosus invertase in
cell extracts was modulated by the ratio of GSH and
GSSG, it was suspected that a change from a reduc-
ing to an oxidizing environment in the hyphae could
oxidize invertase thiol(s) and cause its inactivation.
Several plant and animal enzymes are regulated by
GSH/GSSG ratio [12,13]; however, no other inver-
tase is regulated similarly. The ratio GSH/GSSG
(Table 1) was maximum in mycelia sampled at 6 h
NADP NADPH Ratio NADPH to
(nmol g dry wt31 ) (nmol g dry wt31 ) NADP
20 10 80 17 4.0
29 10 60 17 2.1
18 5 30 7 1.7
45 80 16 1.7
n.d.b n.d. n.d.
30 10 30 6 1.0
44 A. Chaudhuri et al. / FEMS Microbiology Letters 177 (1999) 39^45
when invertase activity was increasing rapidly but
growth was very small, though perceptible. This ra-
tio is consistent with the NADPH/NADP ratio and
the activity of the pentose phosphate pathway (Table
2) which generates reduced coenzyme required for
glutathione reductase catalyzed reaction for GSH
production
(GSSG+NADPH+H C2GSH+NADP ). Although
the GSH/GSSG ratio declined between 6 and 12 h,
the invertase activity reduced after 12 h. Despite the
perturbations, the GSH/GSSG ratio remained V50,
and in fair agreement with that in other organisms
[14]. A cautious view is that the intracellular environ-
ment in T. lanuginosus remained favorable for inver-
tase. Why then did invertase activity show an inverse
correlation with biomass?
A revealing observation was that the time of max-
imum DNA synthesis (0^12 h) was related to the
time of maximal growth rate of the fungus [3], and
to the burst in invertase activity, but not to the time
of maximal increase in biomass (8^16 h) [3]. In fungi,
growth and nuclear division are conned to hyphal
tips [15,16]. The pattern of 3 H-thymidine incorpora-
tion suggested a higher frequency of branch initia-
tion, the number of hyphal tips and of nuclear divi-
sions at the early times. The increase in biomass
mainly results from the deposition of cell wall poly-
saccharides in the elongating hyphal cells [16]. There-
fore, for an enzyme that is localized in the apical
region, rather than uniformly distributed in the hy-
pha (as are invertase in N. crassa and trehalase in T.
lanuginosus), its activity in mycelial samples will ap-
pear to diminish as the apical region is diluted by cell
elongation and wall thickening in the proximal re-
gion of the hypha (Fig. 1). Our attempt to demon-
strate invertase in the hypha by immunouorescence
staining was thwarted by the failure to purify inver-
tase [2]. A sucient quantity of mycelium for enzyme
purication, from the early hours of culture, was
dicult to obtain. The diculty in purifying inver-
tase was exacerbated by the inactivation of enzyme
during the purication steps. However, two other
observations strongly support the view that in T.
lanuginosus invertase is localized in the hyphal tips.
One, the activities of both invertase and a proton-
driven sucrose transporter in T. lanuginosus followed
a parallel course of induction and decline [17]. This
suggests that invertase is restricted to the apical re-
FEMSLE 8879 15-7-99
gion where nutrient transporters have been postu-
lated to be preferentially localized [18]. An associa-
tion of sucrose transporter and invertase would
provide T. lanuginosus a special advantage in scav-
enging and utilizing sucrose from the environment.
Indeed, in mixtures of sucrose and glucose, the fun-
gus utilizes sucrose faster than glucose [3]. Second,
invertase synthesis in T. lanuginosus was dependent
on growth and DNA synthesis [1], events that occur
in the apical region [15,16]. Sucrose is not only nec-
essary for the induced synthesis of sucrose transport-
er and invertase [1,17], but also for maintaining a
reducing intracellular environment for invertase ac-
tivity through the generation of reducing power
(NADPH), which in turn promotes the reduction
of GSSG to GSH. Among the other purposes for
which the reducing power would be required in the
growing hyphal tips are biosynthesis of membrane
fatty acids and the conversion of ribonucleotides to
deoxyribonucleotides for DNA synthesis. It needs to
be emphasized that the measurements of glutathione
and of reduced coenzyme describe the average prop-
erty of mycelial sample, not their concentrations in
the hyphal compartments. It is quite likely that
throughout growth, the hyphal tip has a reducing
environment.
In conclusion, the distinctive pattern of invertase
development in T. lanuginosus is a consequence of a
combination of reasons: invertase being an induced
enzyme; a thiol protein; it being dependent on a
reducing intracellular environment for activity; and
being localized in the hyphal tip. The study suggests
that the activity of some redox-sensitive intracellular
enzymes in fungi may be optimized by their induced
synthesis as required and/or their placement in the
most strategic location in the hypha.
Acknowledgements
This work was supported by Department of Sci-
ence and Technology, Government of India.
References
[1] Maheshwari, R., Balasubramanyam, P.V. and Palanivelu, P.
(1983) Distinctive behaviour of invertase in a thermophilic
 A. Chaudhuri et al. / FEMS Microbiology Letters 177 (1999) 39^45
fungus Thermomyces lanuginosus. Arch. Microbiol. 134, 255^
260.
[2] Chaudhuri, A. and Maheshwari, R. (1996) A novel invertase
from a thermophilic fungus Thermomyces lanuginosus: Its re-
quirement of thiol and protein for activation. Arch. Biochem.
Biophys. 327, 98^106.
[3] Maheshwari, R. and Balasubramanyam, P.V. (1988) Simulta-
neous utilization of glucose and sucrose by thermophilic fungi.
J. Bacteriol. 170, 3274^3280.
[4] Hill, E.P. and Sussman, A.S. (1964) Development of trehalase
and invertase activity in Neurospora. J. Bacteriol. 8, 1556^
1566.
[5] Somogyi, M. (1952) Notes on sugar determination. J. Biol.
Chem. 195, 19^23.
[6] Anderson, M.E. (1985) Determination of glutathione and glu-
tathione disulde in biological samples. Methods Enzymol.
113, 548^555.
[7] Fahey, R.C. and Newton, G.L. (1987) Determination of low-
molecular weight thiols using monobromobimane uorescent
labelling and high performance liquid chromatography. Meth-
ods Enzymol. 143, 85^96.
[8] Klingenberg, M. (1987) Nicotinamide adenine dinucleotides
and dinucleotide phosphates. In: Methods of Enzymatic Anal-
ysis (Bergmeyer, J. and GraMl, M., Eds.), Vol. 7, pp. 251^267.
Verlag-Chemie, Weinheim.
[9] Meister, A. and Anderson, M.E. (1983) Glutathione. Annu.
Rev. Biochem. 52, 711^760.
[10] Marzluf, G.A. and Metzenberg, R.L. (1967) Studies on the
FEMSLE 8879 15-7-99
45
functional signicance of the transmembrane location of in-
vertase in Neurospora crassa. Arch. Biochem. Biophys. 120,
487^496.
[11] Hanks, D.L. and Sussman, A.S. (1969) The relation between
growth, conidiation and trehalase activity in Neurospora cras-
sa. Am. J. Bot. 56, 1152^1159.
[12] Buchanan, B.B. (1991) Regulation of CO2 assimilation in oxy-
genic photosynthesis : The ferredoxin/thioredoxin system.
Arch. Biochem. Biophys. 288, 1^9.
[13] Terada, T., Maeda, H., Okamoto, K., Nishinaka, T. and Miz-
oguchi, T. (1993) Modulation of glutathione S-transferase ac-
tivity by a thiol/disulde exchange reaction and involvement
of thiol transferase. Arch. Biochem. Biophys. 300, 495^500.
[14] Penninckx, M.J. and Elskens, M.T. (1993) Metabolism and
functions of glutathione in microorganisms. Adv. Microbiol.
Physiol. 34, 239^301.
[15] King, S.B. and Alexander, L.J. (1969) Nuclear behaviour,
septation, and hyphal growth of Alternaria solani. Am. J.
Bot. 56, 249^253.
[16] Wessels, J.G.H. (1986) Cell wall synthesis in apical hyphal
growth. Int. Rev. Cytol. 104, 37^79.
[17] Palanivelu, P., Balasubramanyam, P.V. and Maheshwari, R.
(1984) Co-induction of sucrose transport and invertase activ-
ities in a thermophilic fungus Thermomyces lanuginosus. Arch.
Microbiol. 139, 44^47.
[18] Harold, F.M., Kropf, D.L. and Caldwell, J.J. (1985) Why do
fungi drive electric currents through themselves? Exp. Mycol.
9, 183^186.