Ostrich pepsinogens I and II: Purification, activation and chemical and immunochemical characterization of the enzymes from the proventriculus

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Int. J. Biochem. Cell Biol. Vol. 27, No. 6, pp. 613-624, 1995

Pergamon

1357-2725(95)00018-6

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1357-2725/95 $9.50 + 0.00

Ostrich Pepsinogens I and II: Purification, Activation and Chemical and Immunochemical Characterization of the Enzymes from the Proventriculus B R E T T I. P L E T S C H K E , 1 R Y N O J. NAUDI~, 1. W I L L E M O E L O F S E N , I KOJI MURAMOTO, 2 FUMIO YAMAUCHI 2 1Department of Biochemistry, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa and :Department of Applied Biological Chemistry, Faculty of Agriculture, Aoba-Ku, Sendai 981, Japan Pepsins are a series of gastric proteases secreted as inactive precursors (pepsinogens) which are active at acidic pH. The aim of this study was to purify ostrich pepsin(ogen)s and to compare their biochemical and immunological characteristics with those of pepsin(ogen)s of mammalian and avian origin. Ostrich pepsinogens were purified by ammonium sulphate fractionation, Toyopearl Super Q-650S chromatography and rechromatography, and hydroxylapatite chromatography of a pH 8.0 mucosal extract. Pepsins were obtained through acidification, and purified by chromatography on SP-Sephadex C-50. Amino acid compositions, N-terminal sequences, Ouchteriony double-diffusion as well as Western blot analysis were performed. Two pepsinogens were isolated and purified from the proventriculus of the ostrich, pepsinogens I and I1. Both pepsinogens and pepsins were purified to homogeneity as shown by PAGE and SDS-PAGE, with SDS-PAGE revealing Mr values of 40,400 and 41,900 for pepsinogens I and II, respectively. SDS-PAGE revealed Mr values of 36,000 and 36,300 for ostrich pepsins I and II, respectively. Ostrich pepsinogens I and II were found to have identical N-terminal sequences, with Asp as N-terminal amino acid. Amino acid compositions were obtained for both pepsinogens, with ostrich pepsinogen I being slightly smaller in size with a total of 356 residues compared to 371 for ostrich pepsinogen II. Pepsinogen II showed a pI of 4.29. Ostrich pepsinogens I and II were found to be immunologically separate entities, and no cross-reactivity was observed between anti-(ostrich pepsinogen I/II) sera and porcine pepsin/pepsinogen. The study indicates that only two pepsinogens are present in the ostrich. They differ in terms of electrophoretic mobility, molecular mass and immunological reactivity, but have been found to have identical N-terminal sequences. It is concluded that both pepsinogens belong to the pepsinogen A class of aspartyl proteases (EC 3.4.23.1). Keywords: Ostrich

Pepsinogen(s)

Immunochemical Purification

Activation

Int. J. Biochem. Cell Biol. (1995) 27, 613-624

INTRODUCTION Pepsin, an acidic proteolytic enzyme, is found in the stomachs o f almost all vertebrates. The enzyme is stored and secreted in the form o f pepsinogen, its inactive precursor. Pepsinogen is rapidly converted to pepsin in an acidic environment via an autocatalytic process with *To whom correspondence should be addressed.

Abbreviations: BCIP, bromochloroindolyl phosphate; NBT, nitroblue tetrazolium; PEG 20,000, Polyethylene glycol 20,000; PVDF, polyvinylidene diftuoride. Received 27 July 1994; accepted 1 February 1995.

the accompanied removal o f a 42-47 amino acid residue from its N-terminus (Taylor and Tyler, 1986; Foltmann, 1988). Pepsins are characterized by a low p H optim u m (pH 2-3.5), inhibition by pepstatin A, the presence of two catalytic aspartyl residues in the active site, and are proteinases o f b r o a d side-chain specificity. F o r an excellent review on pepsins and other aspartic proteinases, refer to Davies (I 990). Sequence studies have shown a high degree o f h o m o l o g y for the pepsins in the N-terminal domain, Asp32-Thr-Gly-Ser, and Asp2~5-Thr -

613

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Gly-Ser/Thr in the C-terminal domain. Furthermore, this high degree of sequence homology in the active site is not only characteristic for all pepsins, but also for all aspartic proteases of mammalian, plant and fungal origin (Davies, 1990; Fruton, 1987). Compared to pepsinogens of fungal and mammalian origin, literature data on the avian pepsinogens is limited. A detailed investigation on chicken pepsinogen has been reported in terms of isolation and purification, covalent structure, glycosylation pattern, activation mechanism and proteolytic data (Pichovfi and Kostka, 1990; Bohak 1969; Baudys and Kostka, 1983; Baudys et al., 1985; Pichov/L et al., 1985; Donta and Van Vunakis, 1970). Information on other avian pepsinogens has been limited to some knowledge on characteristics of Japanese quail pepsin (Esumi et al., 1980), ostrich pepsinogen (Streicher et al., 1985) and duck pepsin (Pichovfi and Kostka, 1990). The aim of this study was to extend our current knowledge of pepsinogens present in the proventriculus of the ostrich, and the subsequent comparison of their biochemical and immunological characteristics with those of other species, in particular those of mammalian and avian origin.

M A T E R I A L S AND M E T H O D S

Materials Porcine pepsinogen and pepsin, calibration proteins for SDS-PAGE, and bovine haemoglobin were supplied by Sigma Chemical Co., U.S.A. Immobilon-PVDF membrane was purchased from Millipore Corporation (Bedford, Mass.). Bio-Lyte 3-10 carrier ampholytes for isoelectric focussing was supplied by BioRad Laboratories. Toyopearl Super Q-650S was obtained from TOSOH Corp., Japan. The phosphatase-labelled affinity purified goat antirabbit IgG (H + L) and BCIP/NBT phosphatase substrate system was purchased from Kirkegaard and Perry Laboratories, Inc. (Maryland, U.S.A.). Ostrich proventriculi Ostrich (Struthio camelus) proventriculi were excised approx. 20min after the birds were slaughtered at an ostrich abattoir. The proventriculi were separated from the muscular gizzards, washed in saline, kept on ice for +_ 6 hr and stored at -20°C until required.

Isolation and purification Preparation of the mucosal extract. A single frozen proventriculus (883 g) was thawed in a 37°C water bath and the surrounding mucus removed. The central portion of the proventriculus containing the oxyntic glands was excised, and cut into small pieces. All subsequent steps were performed at 4°C. These glands (237 g) were minced, and subsequently homogenised (2.5ml/g) in 0.1M sodium phosphate buffer, pH 8.0 (Waring Blender, 30 sec at high speed). The extract was centrifuged at 14,000g for 20min. The supernatant (S1,425 ml) was adjusted to pH 8.0 with i M NaOH and brought to 10% saturation with solid ammonium sulphate. After 1 hr the supernatant ($2, 400ml) was collected after centrifugation at 14,000 g for 45 min, brought to 45% saturation with solid ammonium sulfate and left overnight. The precipitate was separated from the supernatant ($3, 315 ml) by centrifugation and dissolved in 230 ml of 0.02 M triethanolamine/HC1 buffer, pH 8.0 (dissolving buffer). After dialysis against the dissolving buffer the supernatant ($4, 250 ml) was collected by centrifugation at 14,000g for 15 rain, added to 250 ml Toyopearl Super Q-650S equilibrated in dissolving buffer and stirred overnight. The slurry was allowed to settle, the supernatant poured off, and the resin packed into a column (2.5 x 50 cm). The column was washed extensively with dissolving buffer until the Az80nm had reached baseline. The sample was eluted from the column by a linear salt gradient of 0-0.5 M NaC1 (using two 11 chambers) in dissolving buffer at 220 ml/hr. Fractions (19 ml) were collected and monitored for A280n m and potential proteolytic activity towards bovine haemoglobin (Fig. 1). Five peaks were obtained, with all fractions being proteolytically active. All fractions were concentrated with PEG 20,000. Fractions S4B and $4C represent the minor (pepsinogen I) and major (pepsinogen II) fractions, respectively, in the proventriculus of the ostrich, as identified by SDS-PAGE and high specific activities. Purification of pepsinogen I. Fraction S4B was dialysed against distilled water, and then against 0.5mM potassium phosphate buffer, pH 8.0. The fraction was divided into smaller fractions of _ 80 mg protein each, and loaded onto a BioGel HTP hydroxylapatite column (0.5 x 21 cm), previously equilibrated with 0.5 mM potassium phosphate buffer, pH 8.0.

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FRACTION NO. (19ML/TUBE) Fig. 1. Super Q-650S chromatography of fraction $4. Column dimensions: 2.5 x 50cm. Flow rate: 220ml/hr. The pepsinogens were eluted with a linear salt gradient of 043.5 M NaCI in 0.02 M triethanolamine/HC1 buffer (pH 8.0). (C]), Azs0nm; ( I ) , potential proteolytic activity and ( ), NaC1 concentration.

Stepwise elution by potassium phosphate buffers, p H 8.0, were used in the range of 0.5-400 mM. The peaks were pooled according to potential proteolytic activity and purity on P A G E as three fractions, S4BI, $4B2 and $4B3 [Figs 2 and 4(a)]. The fractions were concen-

trated with P E G 20,000, dialysed extensively against distilled water, and lyophilised. Purification of pepsinogen II. Fraction $4C was dialysed against dissolving buffer over a period of 48 hr. The sample was rechromatographed on a Super Q-650S column (1.5 x

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FRACTION NO. (2MidTUBE) Fig. 2. Hydroxylapatite chromatography of fraction S4B (pepsinogen I). Column dimensions: 0.5 x 21 cm Flow rate: 20 ml/hr, Stepwise elution was performed with potassium phosphate buffers (pH 8.0) in the range of 0.5-400 raM. (--), A2s0,m and (I), potential proteolytic activity.

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FRACTION NO. (10ML/TUBE) Fig. 3. Super Q-650S rechromatography of fraction $4C (pepsinogen II). Column dimensions: 1.5 x 28.5 cm. Flow rate: 220 ml/hr. Pepsinogen II was eluted with a linear salt gradient of 04).5 M NaC1 in 0.02 M triethanolamine/HC1 buffer (pH 8.0). ([3), A~80,m; (11), potential proteolytic activity and (--), NaC1 concentration.

28.5 cm), previously equilibrated with dissolving buffer. A linear salt gradient of 0-0.5 M NaC1 (using two 400 ml chambers) in dissolving buffer was used at a flow rate of 220 ml/hr. Fractions (10 ml) were collected and monitored for A280°m and potential proteolytic activity (Fig. 3). A major peak (fraction $4C1) with potential proteolytic activity eluted, which was divided into four subfractions (S4Cla, S4Clb, S4CIc and S4Cld). The four fractions were pooled, dialysed against distilled water, and concentrated with PEG 20,000. PAGE analysis showed fractions S4Clb and S4Clc to have the highest yield [Fig. 4(b)]. These fractions were subsequently pooled, dialysed exhaustively against distilled water, and lyophilised [fraction S4CI(b + c)].

formic acid buffer, pH 4.0, resulted in the elution of a peak with proteolytic activity towards haemoglobin (Fig. 5). The activation peptide(s) was eluted from the column with the application of a 0.3 M triethanolamine/formic acid buffer, pH 4.0. Pepsin activated in this manner was characterized on SDS-PAGE, pooled accordingly, dialysed against distilled water, and lyophilised. Pepsinogen II was activated as follows: 100mg pepsinogen II was dissolved in 2ml 0.001 M HC1 (pH 3.0), and incubated at 37°C for 10min. The reaction was stopped with the addition of 10 ml 0.02 M triethanolamine/ formic acid buffer, pH 4.0. Pepsin II and activation peptide(s) were separated on SPSephadex C-50 as before.

Activation of pepsinogens I and H

Assays of proteolytic activity Potential proteolytic activity was determined according to a modification of the method described previously (Streicher et al., 1985), essentially based on the method of Anson (1938). The reaction mixture consisted of 0.1 ml zymogen solution acidified by the addition of 0.4ml 0.135M HC1 in order to activate the pepsinogens into their corresponding pepsins. After a 10min incubation period at 37°C, 0.75ml of 1% acid-denatured haemoglobin (Pickup and Hope, 1971) was added, and incu-

Pepsin I was generated from pepsinogen I as follows: 40mg ostrich pepsinogen I was dissolved in 1 ml 0.01 M HC1 (pH 2.0), and incubated at 14°C for 30 min. The reaction was stopped by the addition of 5 ml 0.02 M triethanolamine/formic acid buffer, pH 4.0. Pepsin I and activation peptide(s) were separated on a column (1.6 × 20.5 cm) of SP-Sephadex C-50 (acid-alcohol swollen), equilibrated in 0.02 M triethanolamine/formic acid buffer, pH 4.0, at 20 ml/hr. Elution with 0.02 M triethanolamine/

Ostrich pepsinogens 1 and I1

617

(b) Fig. 4. (a) PAGE patterns of pepsinogen l under nonreducing conditions. The concentration ofacrylamide was 15%, and a Tris/glycine buffer, pH 8.8, was used. Lane 1, ostrich pepsinogen Ik lane 2. ostrich pepsinogen I (previous isolation): lane 3, fraction S4B; lane 4, fraction S4B[, lane 5, fraction $4B2 and lane 6, fraction $4B3. (b) PAGE patterns of pepsinogen II under nonreducing conditions. The concentration of acrylamide was 15%, and a Tris/glycine buffer, pH 8.8, was used. Lane 1, porcine pepsin A: lane 2, ostrich pepsinogen lI (previous isolation): lane 3, fraction $4C1; lane 4, S4CIa; lane 5, S4CIb; lane 6, S4CIc and lane 7, S4CId.

b a t e d for a n o t h e r 1 0 m i n . T h e r e a c t i o n was s t o p p e d by the a d d i t i o n o f l m l 10% T C A . T h e s u s p e n s i o n was c e n t r i f u g e d at 3500 r p m in a B e c k m a n M o d e l T J - 6 c e n t r i f u g e w i t h a s w i n g o u t r o t o r , a n d the a b s o r b a n c e o f T C A s o l u b l e p r o d u c t s at 2 8 0 n m was used as a m e a s u r e o f p r o t e o l y s i s . O n e p r o t e o l y t i c unit was

defined as the a m o u n t o f e n z y m e (in m g p r o t e i n ) w h i c h p r o d u c e d an i n c r e a s e in A280,,, o f 1.0 in 10 m i n u n d e r the a s s a y c o n d i t i o n s .

Protein concentration P r o t e i n c o n c e n t r a t i o n s w e r e d e t e r m i n e d using the m o d i f i e d L o w r y m e t h o d o f P e t e r s o n (1977)

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1.50 -1.25 0.75-1.00 ! -0.75

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F R A C T I O N NO. ( 2 M L / T U B E ) Fig. 5. SP Sephadex C-50 chromatography of the activation of pepsinogen I mixture. Column dimensions: 1.6 x 20.5cm. Flow rate: 20 ml/hr. Pepsin I and activation peptide I were eluted with 0.02 and 0.3 M triethanolamine/formicacid buffer,pH 4,0, respectively.(E]), A280nmand (11), potential proteolytic activity. with bovine serum albumin and commercial porcine pepsinogen as standards. Purity tests and molecular mass determinations

PAGE was performed in 15% gels according to the method of Laemmli (1970) as described in the Bio-Rad Mini-protean T M Dual Slab Cell instruction manual. S D S - P A G E was performed in 12% gels. The following low molecular mass standards were used, with Mr values in parentheses: ~-lactalbumin (14,200), trypsin inhibitor (20,100), trypsinogen (24,000), carbonic anhydrase (29,000), glyceraldehyde 3-phosphate dehydrogenase (36,000), ovalbumin (45,000) and bovine serum albumin (66,000). Protein bands were made visible by staining the gels with Coomassie Brilliant Blue G-250.

Amino acid analysis

Lyophilised fractions were hydrolysed in 4 M methane sulfonic acid in vacuo at l l0°C for 20hr. The hydrolysates were analysed on a Beckman 118BL amino acid analyser according to the method of Spackman et al. (1958). Fractions were also S-carboxamidomethylated, and hydrolysed in 6M HC1 containing 3% phenol at I I0°C for 22 hr (Muramoto and Kamiya, 1990). Amino acid sequence determination

N-terminal sequence analysis was performed by automatic sequencing on a Shimadzu PSQ-1 gas-phase protein sequencer using the fluorescein isothiocyanate (FITC)-phenylisothiocyanate (PITC) double coupling method (Muramoto et al., 1993).

Isoelectric focussing

Isoelectric focussing was performed in the vertical polyacrylamide Bio-Rad minigel system used for PAGE and S D S - P A G E above, according to the method of Robertson et al. (1987). IEF-Mix 3.5-9.3 contained the following standards, with pI values in parentheses: soybean trypsin inhibitor (4.55),/%lactoglobulin A (5.13), human and bovine carbonic anhydrase (6.57 and 5.85, respectively), horse myoglobin (6.67 and 7.16), L-lactate dehydrogenase (8.3 and 8.4) and trypsinogen (9.3).

lmmunochemical studies

Rabbits were injected intramuscularly via the thigh muscle with 2 mg ostrich pepsinogen I or ostrich pepsinogen II dissolved in 0.25ml saline and emulsified with an equal volume of Freund's complete adjuvant. The rabbits were bled two weeks later via the marginal ear vein. A second injection of pepsinogen I/II was performed at week 3. Animals were bled at 5 weeks and 8 weeks. A satisfactory antibody titer was obtained at 5 weeks, as judged by double

Ostrich pepsinogens I and II Table I. Summary of the purification procedure for ostrich pepsinogens I and II Total Total Specific Volume activity protein activity Step and fraction (mI) (U) (g) (U/mg protein) Crude extract (SI) 425 92,888 9.988 9.3 10~45% ammonium sulphate ($4) 250 54,124 2.313 23.4 Super-650 S4B 31.5 9,958 0.260 38.3 $4C 22.5 [7,926 0.231 77.6 Super Q-650 rechromatography (freezedried) $4C1 (b + c) (pepsinogen II) -5,056 0.079 64.0 Hydroxylapatite chromatography (freezedried) S4B1 (pepsinogen I) 64 1,746 0.012 145.5 $4B2 (pepsinogen I) 59.5 [,429 0.005 285.7 $4B3 20 298 0.003 99.4

immunodiffusion according to the Ouchterlony technique described by Johnstone and Thorpe (1982). Precipitin lines were detected with Coomassie Brilliant Blue G250. Immunoblot analysis was performed as set out in the BioRad Mini Trans-Blot electrophoretic transfer cell instruction manual, catalogue numbers 1703930 and 170-3935, with minor modifications. S D S - P A G E was performed under reducing conditions as outlined in the "Materials and Methods" section with 12% acrylamide, and then the protein components were electrotransferred onto Immobilon-PVDF membranes. The membranes were blocked with TBS containing 5% (w/v) BSA, washed and incubated with either rabbit anti-ostrich pepsinogen I serum or rabbit anti-ostrich pepsinogen II serum in 1% (w/v) BSA in TBS, containing 0.05% (v/v) Tween-20. The membranes were washed and incubated with peroxidase-labelled secondary (goat) antibody (diluted 2 0 0 x ) . A BCIP/NBT phosphatase substrate system was used as an effective immunohistochemical staining method. RESULTS AND DISCUSSION After homogenisation of the gastric mucosa and fractionation with ammonium sulphate (10-45% saturation), the supernatant ($4) was chromatographed on a Toyopearl Super Q-650S column as shown in Fig. 1. Five fractions were obtained, all showing potential proteolytic activity towards the haemoglobin substrate. Fractions S4B and $4C represent the two isoforms of ostrich pepsinogen, as detected and identified by PAGE, with fraction $4C being the major pepsinogen in the ostrich proventriculus, namely pepsinogen II. Pepsinogen I is present in smaller quantities as fraction S4B. The three remaining peaks S4A, S4D and S4E were shown

619

Yield 100 58 10.7 19.3 5.4 1.9 1.5 0.3

to contain a substantial amount of contaminating proteins with negligibly small amounts of ostrich pepsinogens present, as revealed by SDS P A G E and their relatively low specific activities. Specific activities of fractions S4B and $4C were determined as 38.3 and 77.6 U/rag protein, respectively (Table 1). During hydroxylapatite chromatography pepsinogen I (fraction S4B) separated into a number of peaks (Fig. 2). The peaks were pooled as three fractions according to potential proteolytic activity and purity on PAGE. Fractions S4BI, $4B2 and $4B3 were recovered with specific activities of 145.5, 285.7 and 99.4 U/rag protein, respectively (Table 1). PAGE and S D S - P A G E showed a single component for fractions S4BI and $4B2 (Fig. 4a), with an Mr of 40,400. Pepsinogen II (fraction $4C) was rechromatographed on Super Q-650S (Fig. 3). A major peak (fraction $4C1) eluted, which was divided into four subfractions. PAGE analysis showed fractions S4CIb and S4Clc to be identical [Fig. 4(b)]. These fractions were pooled and lyophilised. Fraction S4Cla contained contaminating proteins with a small amount of pepsinogen II present (low specific activity), as can be seen from the P A G E pattern [Fig. 4(b)]. Fraction S4CId revealed a single component [Fig. 4(b)], but exhibited a low specific activity. The lyophilised fraction $4Cl (b + c) revealed a specific activity of 65 U/mg protein (Table 1). From the purification table (Table 1) it can be seen that the yield of enzyme and total protein dramatically decreased with ammonium sulphate treatment. Hydroxylapatite chromatography also led to a decrease in yield from 10.7 to 3.7%, but with a subsequent marked increase in specific activity from 38.3 to 99.4-285.7 U/mg protein, which can be attributed to the removal of a large amount of contaminating proteins. Both PAGE and S D S - P A G E showed a single

620

Brett 1. Pletschke et al.

component with an Mr of 41,900 for the major pepsinogen in the ostrich stomach, i.e. for pepsinogen II. From preliminary activation experiments it was decided to activate ostrich pepsinogens I and II at pH 2.0 and 14°C for 30 min, and at pH 3.0 and 37°C for 10 min, respectively. Under these conditions, the activation of the pepsinogens to their respective pepsins proceeded to completion with a minimal degree of autolysis. Both pepsins showed pH optima of 2.0, with pepsins I and II showing specific activities of 108 and 45.3 U/mg protein, respectively. Because of the activity of pepsinogen I at pH 2.0, the temperature was lowered to 14°C to slow the reaction and to minimise autolysis. A typical elution profile on SP-Sephadex C-50 after activation is shown in Fig. 5. The pepsin, being cationic in character, was eluted with a 0.02 M triethanolamine/formic acid buffer, pH 4.0. As the activation peptide was cationic, a higher salt concentration (0.3M triethanolamine/formic acid buffer, pH 4.0) was required for its elution. Pepsinogen II revealed a pI of 4.29, a value close to that of 3.7 reported for porcine pepsinogen (Fruton, 1987), while the pI of pepsinogen I could not be determined in the pH range 3.5-9.3. Data relating to isoelectric points in the literature is extremely limited. The determi-

nation of isoelectric points of aspartyl proteases has rarely been attempted due to the intrinsic problems involved in working with an enzyme with such a low pI. Ostrich pepsinogens were found to be stable at pH 8.0, and no autocatalytic conversion of pepsinogens to their respective active forms was detected. Donta and Van Vunakis (1970) reported that chicken pepsinogens are not very stable upon storage at pH 7.5, and are autocatalytically converted into their respective pepsins. Lyophilisation of chicken pepsinogens also resulted in a loss of potential peptic activity. Both ostrich pepsinogens I and II were found to be stable upon lyophilisation, and no loss in potential proteolytic activity was detected. Amino acid composition results obtained for ostrich pepsinogens I and II are compared to those of other species, and are presented in Table 2. All values reported in Table 2 result from acid hydrolysis. Accurate results for the amino acid compositions for ostrich pepsins I and II, as well as their activation peptides, could not be obtained. The activation peptides were no longer intact and autolysis of the pepsins occurred. The high ratio of acidic amino acids, i.e. Asp and Glu (assuming a low content of Asn and Gin) as compared to basic amino acids is characteristic of pepsinogens and the rest of the

Table 2. The amino acid composition (molar ratios) of ostrich pepsinogens in comparison with pepsinogens A derived from other species Residue

Ostrich I a

Asx Thr Ser Glx Pro Cyh Gly Ala Val Met lie Leu Tyr Phe His Lys Trp Arg

31.2 22.8 29.5 41.6 19.1 3.7 38.0 20.4 19.7 6.4 20.0 30.1 19.6 19.1 5.4 11.9 6.1 8.6

(31) (23) (30) (42) (19) (6) f (38) (20) (20) (6) (20) (30) (20) (19) (5) (12) (6) (9)

Ostrich II a 42.1 21.7 46.5 29.7 22.7 5.1 35.0 20.1 20.0 8.7 21.9 33.4 19.4 20.4 6.1 8.0 5.3 5.0

(42) (22) (46) (30) (23) (6) f (35) (20) (20) (9) (22) (33) (19) (20) (6) (8) (5) (5)

Chicken b

Dogfishc

Duck d

Porcine e

39 26 41 25 18 7 31 17 25 9 23 28 22 20 8 17 4 7

44 23 43 39 19 7 40 18 23 7 22 28 19 17 7 14 5 14

31 26 39 37 26 6 32 25 25 6 21 31 15 18 5 16 4 I1

46 28 46 28 18 6 36 20 25 4 26 34 16 16 3 10 5 4

Total

356

371

367

389

374

371

Mmin

39,167

40,149

40,425

42,575

40,511

39,659

aAverage of duplicate values. bBaudys and Kostka (1983). CBar-Eli and Merrett (1970). dpichov~i and Kostka (1990). eKageyama and Takahashi (1986). fAssumed value of 6 to account for 3 disulfide bridges.

Ostrich pepsinogens I and II

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L

R

Q

N

L

I

K

L

V

K

L

V

R

Bovine A"

S

V

V

K

L

V

K

K

K

S

L

R

Q

N

L

I

E

Ostrich II

D

V

S

K

L

R

K

G

K

S

L

R

K

H

L

K

D

Ostrich 1

D

V

S

KI

L

R

K

G

K

S

L

R

Porcine A"

D E H

X

L

E

X

Fig. 6. Comparison of the N-terminal sequences of pepsinogens A from various species and ostrich pepsinogens I and II. Residues that are in boxes are those common to pepsinogens A of various species. Shaded residues in boxes are residues that do not conform to the high degree of homology in the boxed areas. The numbering system is based on the sequence of bovine prochymosin, and hyphens indicate deletions. X denotes unknown residues. *Tanji et al. (1988). aspartyl proteases. Pepsin is dependent on this characteristically high content of Asp and Glu residues for its highly negative character in an electric field, its stability at low p H values, and its low pI (Andreeva and James, 1991). Ostrich pepsinogen I was found to be slightly smaller in size with a total of 356 residues compared to 371 for ostrich pepsinogen II. Ostrich pepsinogen II had a higher content of Asx, Ser, Pro, Met, Ile and Leu residues, with ostrich pepsinogen I being higher in Glx, Gly, Lys and Arg. Similar values were obtained for the other amino acids. Compared to chicken pepsinogen, ostrich pepsinogens I and 1I had a higher content of Glx, Gly, Leu and Trp, but lower values were observed for Thr, Cyh, Val, His and Lys. Streicher e t al. (1985) reported the presence of three pepsinogens in the ostrich and their amino acid composition results appeared to be a mixture of those of ostrich pepsinogens I and II reported in the present study. Other investigators also reported controversial results with pepsinogens. For chicken pepsinogens Bohak (1969) has proposed that this may be due to the fact that the same zymogen may be contaminated with vary-

V

L

T

A

T

Human" Bear"

A

V

E

S

Y

E

y

M

D

A

V

D

E

Q

P

L

E

N

Y

L

D

M

E

Y

F

Q

P

L

E

N

Y

M

D

M

E

Y

F

L

E

N

Y

L

D

T

E

Y

F

N

Y

L

D

T

E

Y

F

N

Y

M

D

S

E

Y

F

M

D

M

A

T

Pig"

I

G

D

E

P

Bovine"

V

S

E

Q

P

L

T

V

S

S

E

P

L

A

T

A

Y

E

Ostrich II Ostrich I

A

G

20

15

10

1

Chicken +

ing degrees of impurities that cochromatograph with them, which may result in the observation that there may be more than one zymogen present. Molecular mass determination from amino acid analysis results gave Mmin values of 39,167 and 40,149 for pepsinogen I and II respectively, which is in agreement with the M r values obtained in S D S - P A G E experiments (40,400 and 41,900, respectively). S D S - P A G E experiments revealed Mr values of 36,000 and 36,300 for ostrich pepsins I and II, respectively. The N-terminal sequence information of ostrich pepsinogens I and I1 showed a high degree of homology between the ostrich pepsinogens and pepsinogen A from other species (Fig. 6). It appears that the ostrich pepsinogens are members of the pepsinogen A class (EC 3.4.23.1). Ostrich pepsinogens I and I1 are identical in N-terminal sequence, when comparing the first 14 amino acids, and are believed to be closely related phylogenetically. N-terminal sequence information of ostrich pepsins I and II also showed homology with those of other species (Fig. 7). It is also evident that the

E

Fig. 7. Comparison of the N-terminal sequences of pepsins from various species and ostrich pepsins I and II. Residues in boxes are those common to pepsins of various species. Shaded residues in boxes are residues that do not conform to the high degree of homology in the boxed areas. The numbering system is based on the sequences of chicken pepsin, and hyphens indicate deletions. *Kageyama et al. (1983); fPichov~. and Kostka (1990).

622

Brett I. Pletschke et al.

Fig. 8. (a) Immuno double-diffusion analysis of pepsinogens. The centre well contained anti-(ostrich pepsinogen 1) serum. The surrounding wells contained (1), saline control: (2), porcine pepsinogen; (3), porcine pepsin: (4), ostrich pepsinogen 1I and (5), ostrich pepsinogen I. Incubation was for 36 hr at 37' C. (b) lmmuno double-diffusion analysis of pepsinogens. The centre well contained anti-(ostrich pepsinogen lI) serum. The surrounding wells contained ( 1). saline control: (2), porcine pepsinogen; (3), porcine pepsin; (4). oslrich pepsinogen II and (5), oslrich pepsinogen 1. Incubation was for 36hr at 37 C.

Ostrich pepsinogens 1 and II N - t e r m i n u s of ostrich pepsin 1I is extended by two a m i n o acid residues as c o m p a r e d to pepsin I. A n t i b o d i e s prepared from rabbits by injection of either purified ostrich pepsinogens I or II did not cross-react with commercial porcine pepsin o g e n or porcine pepsin [Figs 8(a) a n d 8(b)]. This is in line with evidence that the ostrich, being a m e m b e r of the avian species, is far removed from m a m m a l i a n species phylogenetically. N o cross-reactivity occurred between ostrich pepsinogen I a n d the anti-(ostrich pepsin o g e n II) serum [Fig. 8(b)]. Similarly, the crossreaction of ostrich pepsinogen II with anti-(ostrich pepsinogen I) serum was limited to a very faint b a n d in Western Blot analysis, p r o b a b l y as a result of a c o m m o n antigenic d e t e r m i n a n t situated in the almost identical N - t e r m i n a l region o f the two ostrich pepsinogens. Western Blot analyses also showed that the antibodies raised against ostrich pepsinogens I a n d II reacted with the c o r r e s p o n d i n g pepsins, i.e. anti-(ostrich pepsinogen I) serum reacted with ostrich pepsinogen I, as well as with ostrich pepsin I, while anti-(ostrich pepsin o g e n II) serum reacted with ostrich pepsinogen II a n d ostrich pepsin II. F r o m these observations, the c o n c l u s i o n can be d r a w n that the two isoforms of ostrich pepsinogen are imm u n o l o g i c a l l y distinct from one another, a n d that a small antigenic d e t e r m i n a n t region in the N - t e r m i n a l region (i.e. the activation peptide) m a y be shared by ostrich pepsinogens I a n d II. Esumi et al. (1980) reported that Japanese quail pepsinogen a n d chicken pepsinogen C of D o n t a a n d Van V u n a k i s (1970) both crossreacted with a n t i s e r u m to pepsinogen of the other species. However, chicken pepsinogens A a n d D did not cross-react with either chicken pepsinogen C or quail pepsinogen (Esumi et al., 1980). Acknowledgements~he authors gratefully acknowledge

financial support provided by the Foundation for Research Development and the University of Port Elizabeth. We are also indebted to the Klein Karoo Agricultural Co-operation at Oudtshoorn, South Africa, for their generous supply of experimental material. REFERENCES

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Pickup J. C. and Hope D. B. (1971) Protease and ribonuclease activities in bovine pituitary lobes. Biochem. J. 123, 153-162. Robertson E. F., Donnelly H. K., Malloy P. J. and Reeves H. C. (1987) Rapid isoelectric focussing in a vertical polyacrylamide minigel system. Analyt. Biochem. 167, 290-294. Spackman D. H., Stein W. H. and Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1191~1206.

Streicher E., Naud~ R. J. and Oelofsen W. (1985) The isolation and characterization of pepsinogens from the proventriculus of the ostrich Struthio camelus. Comp. biochem. Physiol. 82, 67-72. Tanji M., Kageyama T. and Takahashi K. (1988) Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences. Eur. J. Biochem. 177, 251-259. Taylor P. M. and Tyler M. J. (1986) Pepsin in the toad Bufa Marinus. Comp. biochem. Physiol. 84, 669~572.