Comp. Biochem. PhysioL Vol. 105B,Nos 3/4, pp. 591-595, 1993 0305-0491/93 $6.00+ 0.

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Printed in Great Britain Pergamon Press Ltd

THE ISOLATION A N D PARTIAL CHARACTERIZATION

OF ENTEROKINASE FROM OSTRICH (STRUTHIO
CAMELUS) D U O D E N A L MUCOSA
RYNO J. NAUDI~, D~NIS DA SILVA, WENDY EDGE and WILLEMOELOFSEN
Department of Biochemistry, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000,
South Africa (Fax 041 5042-574)

(Received 3 December 1992; accepted 15 January 1993)

Abstract--1. Detergent-solubilized ostrich duodenal enterokinase was purified by salt fractionation,

DEAE--cellulose and Toyopearl HW-65F chromatography.
2. Gradient gel electrophoresis and SDS-PAGE revealed M r values of 127,000 and 62,200, respectively.
The ostrich enzyme can be assumed to be a dimer.
3. The amino acid composition of ostrich enterokinase is similar to that of mammalian enterokinase.
4. The highest rate of activation of bovine trypsinogen by ostrich enterokinase was at pH 5.2-5.7.

INTRODUCTION Ostrich material

Enterokinase (EC 3.4.4.8.) is located in the brush
border membrane of the duodenum. It plays a key
role in the digestion of proteins as it activates pancre-
atic trypsinogen to trypsin which, in turn, will acti-
vate the other pancreatic zymogens (Anderson et aL,
1977).
Enterokinase has previously been isolated from the
pig (Baratti et al., 1973; Maroux et al., 1971), the cow
(Anderson et al., 1977; Fonseca and Light, 1983;
Jacob and Pattabiraman, 1984), and the human
(Grant and Hermon-Taylor, 1975; Antonowicz et al.,
1980).
Different forms of ostrich trypsin and chymotrypsi-
nogen (Hartley et al., 1987; Van der Westhuizen
et al., 1989; Smith et al., 1992) and a proelastase
(Sutherland et al., 1991) were isolated and purified
from the pancreas of the ostrich. Very little is known
about avian enterokinase and this study was under-
taken to compare the enterokinase of a primitive bird
with that of mammals.
MATERIALSANO METHODS
Chemicals
Porcine enterokinase, DMSO, APNA, bovine
trypsinogen and calibration proteins for SDS-PAGE,
isoelectric focusing and gradient gel electrophoresis
were supplied by Sigma Chemical Co. (St Louis,
MO). BAPNA was obtained from Boehringer-
Mannheim GmbH (Mannheim, Germany) and Serva
Blue G from Serva (Heidelberg, Germany). All other
chemicals were of the highest analytical grade and
were used without further purification.
Abbreviations--BAPNA, ~ -benzoyl-D,L-arginine-4-nitro-
anilide; DMSO, dimethyl sulphoxide; APNA, L-alanine-
4-nitroanilide.
Ostrich duodena were excised _ 20 min after the
birds were killed at the ostrich abattoir. The duodenal
contents were gently squeezed out and the duodena
were cut open. The mucosa was scraped off, kept on
ice for +_6hr and stored at - 2 0 ° C until required.
Isolation and purification
All steps were performed at 4°C. Frozen mucosa
(2.432 kg) was thawed overnight at 4°C and 51 of a
0.01 M Tris-HC1 buffer (pH8.0), containing 1%
Triton X-100 was added and stirred for 5 min.
The diluted mucosal suspension was homogenized
in a Waring blender for 10sec at low speed and
stirred for 2 hr. Solid (NH4)2SO4 was added to 30%
saturation, left to stand for 2 hr and centrifuged at
13,700g for 45min. The supernatant was filtered
through cheesecloth, brought to 70% saturation with
solid ('NH4)2SO 4 and left overnight. The precipitate
was collected by centrifugation, suspended in 1800 ml
distilled water and dialysed against 2001 of running
distilled water. The pH of the retentate was adjusted
to 5.0 and centrifuged at 13,700g for 45min. The
supernatant (fraction 1) was added to 1000ml of
DEAE-cellulose equilibrated in 0.01 M Na-acetate
buffer (pH 5) and stirred for 2 hr. The slurry was
allowed to settle overnight, the supernatant was
poured off and the resin was packed into a column
(5 x 55 era). The column was washed extensively with
0.01 M Na-acetate buffer (pH 5) until the A2s0m of
the effluent was down to a constant low level. The
sample was eluted with 0.01 M Na-acetate buffer
containing 0.12M NaC1 (pH 5) at 200ml/hr. Two
activity peaks emerged, were pooled accordingly,
dialysed free of salt and lyophilised (Fig. 1).
Fraction 1A was applied to a Toyopearl HW-65F
column ( 2 . 6 x 7 2 c m ) , equilibrated with 0.01M
Tris-acetate buffer containing 0.5 M NaC1 (pH 7)

591
592 RYNo J. NAUD~ et al.

2.5 6.0

-5.0
2.0-

Enteroklne~e
-4,0

E,.oj
.~
1.5- (~tA410nrrl/mln x 10 3 )

12

1.0-

2.0

0.5-
-1.0

O.C ~ 0.0
75 i~o 1~5 150 175
Tube no, (15ml/tube)

Fig. 1. DEAE-celluiose chromatography of an enterokinase-active ostrich duodenal mucosa extract.

Column dimensions: 5 x 55 cm. Flow rate: 200 ml/hr. Column was eluted with 0.01 M Na-acetate buffer
(pH 5) containing 0.12 M NaC1. A , enterokinase activity; I-3, aminopeptidase activity.

(running buffer). Fractions were eluted with the
running buffer. SDS-PAGE of the eluted fractions
showed three components which were pooled,
dialysed free of salt and lyophilised (Fig. 2).
Enzyme assays
Enterokinase activity was measured by its activat-
ing effect on bovine trypsinogen. The trypsin formed
was assayed following the addition of the synthetic
substrate BAPNA by the method of Erlanger et al.
(1961). Thirty-three microlitres of bovine trypsinogen
and 33/zl enterokinase were added to 267/~1 of a
40 mM Tris-acetate buffer (pH 5.8) containing 1 mM
CaCI2 and incubated for 30 min at 37°C. At zero
time, 667/zl of the substrate solution (21.8mg
BAPNA dissolved in 3 ml DMSO and made up
to 50 ml with 0.05 M Tris-HCl buffer, pH 8.2) con-
taining 10mM CaCI2 was added. The progress of
the reaction was followed at 410 nm. Enterokinase
blanks (no trypsinogen) and trypsinogen blanks

1.8

1.6

1.4

1.2
E
¢o 1

0.8

0.6

0.4

0.2

0
J
0 20 40 60 80

TUBE no. (5.Sml/tube)

Fig. 2. Toyopcarl HW-65F chromatography of fraction IA. Column dimensions: 2.6 x 72 cm. Flow rate:
130 ml/hr. Column was elutcd with 0.01 M Tris-acctate buffer (pH 7.0) containing 0.5 M NaC1.
 Ostrich enterokinase
(no enterokinase) were also run for each enzyme
dilution and both blank values were subtracted from
the test value before AA410m/min values could be
determined.
From linear portions of the progress curves,
AA410nm/min values were calculated and plotted
against enzyme protein concentration. Regression
lines were calculated. The resultant slopes served as
a measure of specific activity (AA4~om/ndn/mg Serva
protein).
Aminopeptidase activities were measured with
the synthetic substrate APNA (1 raM) which, on
hydrolysis, gave the yellow p-nitroaniline product
(Baratti et al., 1973).
Protein determination
Protein concentrations were determined by using
the modified version of the Read and Northcote
(1981) method. Bovine serum albumin and Scrva
Blue G were used as standard and dye reagent,
respectively.
SDS-PAGE
Gel slabs were prepared (7.5% acrylamide) accord-
ing to the method of Laemmli (1970) as described
in the Bio-Rad Mini-protein ~ II Dual Slab Cell
instruction manual.
The molecular weight standard proteins used, with
Mr values in parenthesis, were: egg albumin (45,000);
bovine serum albumin (66,000); phosphorylase b
(97,000); fl-galactosidase (116,000); and myosin
(205,000).
Polyacrylamide gel-isoelectric focusing
P A G - I E F was performed in gel slabs (250 x 120 x
0.5mm) in the LKB 2117 Multiphor II electro-
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20 I I
4.7 4.9
Fig. 3. Calibration curve for standard proteins on gradient gel electrophoresis. Relative migration (Rf)
vs log Mr was established using (1) BSA (monomer), (2) fl-galactosidase, (3) BSA (dimer) and
(4) catalase. Stars indicate the Rf values of ostrich enterokinase (fraction IA2).
593
Table 1. Summary of purification procedure
Enterokinasv-spccific activity
Step Fraction (AA,0=/rain/rag protein)
DEAE--cellulose IA 48.53 x 10 -2
IB 10.95 x 10-2
Toyopearl HW-65F IAI 10.82
IA2 31.58
1A 3 11.63
phoresis unit, using a LKB constant power supply.
Gels were polymerized in a LKB 2117-200 Ultra-
mould gel casting unit on a sheet of cellophane to
facilitate easy handling of the gel (G6rg et al., 1977).
Gels contained 7.35% acrylamide and 2% ampholine
(pH 3-10). After prefocusing for 30 rain, 10 #g Serva
protein was loaded and focused for 3-3.5hr at
1050 V. Gels were stained with Coomassie Brilliant
Blue G-250 (Blakesley and Boezi, 1977). Marker
proteins, with well-defined pI values in parenthesis,
were: amyloglucosidase (3.55); trypsin inhibitor
(4.55); fl-lactoglobulin A (5.13); carbonic anhydrase
B (bovine) (5.85); carbonic anhydrase B (human)
(6.57); myoglobin (6.76); and trypsinogen (9.30).
Gradient gel electrophoresis
Gradient gel electrophoresis was used to determine
the molecular weight of the intact enzyme. The
5-20% gel was cast in the vertical slab gel electro-
phoresis unit SE 600 from Hoefer Scientific Instru-
ments (San Francisco, CA), using a gradient mixer
(Dunbar et al., 1990). The molecular weight standard
proteins used, with M r values in parenthesis, were:
chymotrypsin (25,000), bovine serum albumin
(66,000), fl-galactosidase (116,000) and catalase
(240,000).
I I 1 I I
,5.1 5.3 ..5.5
Log Mr
594 RYNO J. NAUD~ et al.

Table 2. Amino acid composition (molar ratios) of ostrich (fraction analyser according to the method of Spackman et al.
IA2), pig, cow and human enterokinase
(1958).
Residue Ostrich* Pigt Cow:~ Human§
Asx 110.1)6 (110.0) 130.2 110.7 165.0 Effect of pH
Thr 46.58 (47.0) 72.2 56.8 89.0
Ser 49.82 (50.0) 84.4 78.0 99.0 The effect of pH on the activation of bovine
Glx 147.24(147.0) 124.4 102.8 129.0 trypsinogen by ostrich enterokinase (fraction
Pro 61.48 (61.0) 66.5 52.0 93.0 1A2) was tested at 37°C. The buffers used were
Cyh 41.77 (42.0) 44.9 29.4 24.0
Gly 75.96 (76.0) 86.2 80.4 117.0 50mM Na-acetate (pH4-5.5), 50mM Mes buffer
Ala 75.15 (75.0) 63.9 47.2 71.0 (pH 5.5-6.5) and 50 mM Tris-HCl buffer (pH 6.5-8).
Val 64.19 (64.0) 65.0 50.8 74.0
Met 28.36 (28.0) 16.0 12.6 8.0
All buffers contained 10 mM CaC12.
lie 58.01 (58.0) 56.1 52.6 62.0
Leu 89.79 (90.0) 91.3 72.6 100.0 Carbohydrate content
Tyr 38.29 (38.0) 36.2 30.3 32.0
Phe 45.89 (46.0) 54.3 41.7 58.0 The sialic acid determination was carried out
His 31.27 (31.0) 18.5 15.8 23.0 according to Warren (1959) with an internal zero
Lys 86.40 (86.0) 39.8 36.3 58.0 calibration at 590 nm before monitoring at 549 nm.
Trp 5.29 (5.0) 12.0 11.6 ND
Arg 39.48 (39.0) 29.5 24.8 35.0 The hexose (mannose:galactose---1:1), fucose and
No. of
hexosamine contents were determined by the
residues 1093 1091 908 1237 methods described by Winzler (1955). The principle
Minin 122,521 127,264 92,800 127,280 of internal zero calibration was also used for hexose
% Sugar 4.07 35.0 36.0 57.0
Intact (640 nm zero calibration for monitoring at 540 nm)
tool. wt 127,000 195.790 145,000 296,000 and hexosamine (600 nm zero calibration for moni-
pl 4.58, 4.69 3.99, 4.1 4.69 4.78, 4.9
4.21 toring at 530 nm).
ND: not determined.
*Values for amino acid composition are averages of four determi- RESULTS AND DISCUSSION
nations.
fBarratti et al. (1973).
~Liepnicks and Light (1979). After extraction of the duodenal mucosa and
~Grant and Hermon-Taylor (1975). fractionation with (NH4)2SO4 (30-70% saturation),
the precipitate was dissolved and chromatographer
Amino acid analysis on a DEAE-cellulose column as shown in Fig. 1.
Amino acid analysis was performed on lyophilized Two fractions showing enterokinase and aminopepti-
fraction 1A2. Samples were hydrolysed in evacuated dase activities were obtained (1A and IB), dialysed
sealed tubes in 5.7 N constant boiling HC1 and 10/~1 free of salt and freeze-dried. Fraction IA showed
5% (w/v) phenol for 20 hr at I10°C. Ser and Thr the highest enterokinase-specific activity (Table 1)
values were corrected for hydrolytic losses of 10 and and was chromatographed on a Toyopearl HW-65F
8%, respectively. Trp was determined by hydrolysis column (Fig. 2). SDS-PAGE revealed the presence
in 200 #1 4M MSA (Moore, 1972). The hydrolysates of three components; tubes were pooled as indicated,
were examined on a Beckman 118 BL amino acid dialysed free of salt and freeze-dried. Fraction

0.011

0.01

0.009

0.008
._~
E
~" 0.007

7,
,~ 0.006

0.005

0.004

0.003
4 5 6 7
pH

Fig. 4. Effect of pH on the activation of porcine trypsinogen by ostrich enterokinas¢ (fraction 1A2).
 Ostrich enterokinase
1A2 showed the greatest increase in specific activity
(Table 1).
On SDS-PAGE, one major and one very minor
band were observed for fraction 1A2. The Mr cor-
responding to the major band was 62,200 and that
of the very minor band, 38,400. From gradient gel
electrophoresis, a major band corresponding to Mr
58,850 and a major/minor band of Mr 127,000
were obtained for fraction IA2 (Fig. 3). No other
Mr components were detected and it can be assumed
that the intact enzyme is a dimer. Liepnicks and
Light (1979) showed that mercaptoethanol treatment
cleaved the native enzyme into heavy and light sub-
units of size 115,000 and 35,000Mr, respectively.
On the other hand, Anderson et al. (1977) reported
the subunit sizes to be 82,000 and 57,000. The native
enzyme is made up of two subunits of different
sizes linked by S-S bridges. At least one of the
subunits could, in turn, be made up of smaller
peptides linked by disulphide linkages. Differences in
the conditions of reduction may be responsible for
the selective cleavage of S-S bonds resulting in the
formation of a number of peptide fragments (Jacob
and Pattabiraman, 1984).
Amino acid composition of ostrich enterokinase
is compared to those of other species (Table 2).
Increased values were obtained for Glx, Met, His and
Lys, while Thr, Ser and Trp showed decreased values.
Isoelectric focusing showed two components with pI
values of 4.58 and 4.69. The isoforms of the ostrich
enzyme compare favourably with those of the human
enzyme (Table 2). The total carbohydrate content of
the ostrich enzyme was low (1.01% hexosamine,
2.17% hexose, 0.36% sialic acid and 0.53% fucose)
in comparison with porcine, bovine and human
enterokinase (Table 2).
From Fig. 4 it is evident that the highest rate of
activation of bovine trypsinogen by ostrich entero-
kinase occurred at pH 5.2-5.7. The pH optimum
between 5.5 and 6.0 corresponds with that of other
enzymes.
Acknowledgements--The authors gratefully acknowledge
the financial support granted by the Foundation for Re-
search Development and the University of Port Elizabeth,
and would also like to express their gratitude to the Klein
Karoo Agricultural Co-Operative at Oudtshoorn, South
Africa, for the experimental materials. We thank Francois
van Jaarsveld for the carbohydrate analyses.
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