Identification and characterization of eight porcine pancreatic proteinases, car☐ypeptidase A and amylase after electrophoretic separation using specific substrates

Int. J. Biochera. Vol. 19, No. 7, pp. 633~539, 1987 Printed in Great Britain.All rights reserved

0020-71IX/87 $3.00+0.00 Copyright © 1987PergamonJournals Ltd

IDENTIFICATION A N D CHARACTERIZATION OF EIGHT PORCINE PANCREATIC PROTEINASES, CARBOXYPEPTIDASE A A N D AMYLASE AFTER ELECTROPHORETIC SEPARATION USING SPECIFIC SUBSTRATES BERTIL G. OHLSSON, BJORN R. WESTROMand BORIE W. KARLSSON Department of Zoophysiology, University of Lund, Helgonav~igen 3 B, S-223 62 Lund, Sweden [Tel. (046) 10-9350]

(Received 23 September 1986)

Abstract--1. Porcine pancreatic hydrolases in juice and homogenate surveyed by electrophoretic separation in agarose gel, at pH 8.6 and subsequently characterized using substrates of various specificity, either directly in the gel or after transfer to nitrocellulose (enzymoblotting) showed: 2. Anodal and cathodal trypsin with Bz-Arg-pNA. 3. Chymotrypsin A, B, and C with similar, but not identical, activities to Suc-Ala-Ala-Pro-Phe-pNA, Bz-Tyr-pNA, Suc-Phe-pNA and Ac-Phe-flNE and with differences in their molecular weights and electrophoretical charges. 4. Elastase I and protease E with Suc-(Ala)3-pNA and MeO-Suc-Ala-Ala-Pro--Val-pNA and elastase I also with elastin. 5. Elastase II with the chymotrypsin substrates and with elastin. 6. Carboxypeptidase A with CN-Phe. 7. Amylase with blue starch polymer.

INTRODUCTION

The porcine pancreatic hydrolases have attained growing interest during the years both from the biochemical and physiological points of view, as well as in applied pig research. Several of these hydrolases and their various isoenzyme forms have been isolated and characterized (Gertler et aL, 1977; Ardelt, 1974; Vered et al., 1985; Kobayashi et aL, 1978; Desnuelle et aL, 1970; Gratecos et al., 1969; Voytek and Gjessing, 1971; Travis and Liener, 1965; Martinez et al., 1981; Folk and Schirmer, 1963; Cozzone et aL, 1970) and changes in enzyme activities have been estimated during different nutritional states and during ontogeny (Just et al., 1985; Corring et al., 1978). Usually only a single or a few enzymes at a time have been studied but these studies have not taken into consideration the number and diversity of procine pancreatic hydrolases. Hitherto no experimental overview has been made of the pancreas hydrolases by identifying and characterizing them using substrate specificities. In a mixture of proteinases, such as pancreas homogenate or juice, it is very difficult to state to what extent a substrate is specific or sensitive for the various proteinases or isoenzymes except when sufficient purification and characterization has been performed. The synthetic amino acid and peptide substrates available containing para-nitroanilide as a terminal group exhibit great variance in amino acid compositions, which makes them versatile for specific enzyme analyses. Such substrates can be used both in spectrophotometrical assays (Nagel et al., 1965) and recently also for detection of enzyme activities after electrophoretic separation using enzymoblotting

(Ohlsson et al., 1986). This makes it possible to investigate directly even complex mixtures of proteinases without elaborate purification steps. In this study we have identified porcine pancretic hydrolases and their isoenzyme forms in pancreas homogenate and juice. Localization and characterization of the enzyme activity was achieved using specific substrates after electrophoretic separation. Twelve different substrates with varying degrees of specificity and sensitivity for trypsin, chymotrypsin, elastase, carboxypeptidase A and amylase were used; 9 synthetic low-molecular weight amino acid/peptide substrates and 3 "natural" high-molecular weight substrates. MATERIALS AND METHODS

The various synthetic substrates used were purchased or synthezised (Table I). BOC-Ala-AIa-Pro-OH was built up from the N-terminal end by condensation of the amino acid residues in solution, using N,N-dicyclohexylcarbodiimide/lhydroxy-benzotriazole dissolved in N,N-dimethylformamide as reagents. The amino-group of AlaI was blocked by the introduction of the tert-butyl-oxycarbonyl(BOC)-group (Serva, Heidelberg, F.R.G.). For temporary protection of the carboxy groups, the methyl esters of the amino acids were used (Serva). BOC-Phe-OH was coupled to para-nitroaniline in presence of PC13 (Kasafirek et al., 1976; Goldschmidt and Rosculet, 1960), and the product formed was deprotected, followed by condensation with BOC-Ala-Ala-Pro-OH. In a last step, BOC-AIa-AIa-Pro--Phe-pNA, was deprotected and reacted with succinic anhydride to yield the final substrate. Pancreas homogenate was obtained by homogenization of pancreas from slaughtered pigs (Sus scrofa) in ice-cold 0.2 M Tris-HC1 buffer + 0.05 M CaC12, pH 8.3 (+2°C), in

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Pig pancreatic hydrolases the ratio 1:4 (w/v). The homogenate was centrifuged at 30,000g for I hr at +4°C and the supernatant was saved. Freeze-dried pancreas juice (a generous gift from Dr T. Corring, Lab. Physiol. Nutr., INRA-CNRZ, Jouy-en-Josas, France) and pancreatin (Sigma, St Louis, Mo. U.S.A.) were dissolved in 0.9% NaCI, 50 mg/ml. The homogenate and the juice were activated by incubation with either enteropeptidase or trypsin (1.0 mg/ml 0.9% NaC1) in the ratio 4:1 for 30 min at 37°C. (Ohlsson et al., 1986). Electrophoretic localization o f enzyme activity Electrophoretic separation of the pancreatic hydrolases was performed in 1% agarose gel (HSB, Litex, Glostrup, Denmark) in 0.037 M Ca-Veronal buffer, pH 8.6, on a plastic support (Gel-Bond, Marine Colloids, Rockland, Me, U.S.A.) at 20V/cm (Johansson, 1972). For comparison purposes bromphenol blue-stained porcine serum albumin was used as a marker (endpoint migration 3.5 cm from the application site). After electrophoresis, the gel plate was protein-stained with a silver staining method (Willoughby and Lambert, 1983), or stained for enzyme activity (Table 1), either directly in the agarose gel (Starkey and Barrett, 1976; Borulf et aL, 1979) or by enzymoblotting (Ohlsson et al., 1986). In the latter method, the enzymes were transferred to a nitrocellulose membrane (Trans-Blot, Bio-Rad, Richmond, Calif., U.S.A.) before incubation with various paranitroanilide substrates. For localizing enzyme activities directly in the agarose gel, the gel was incubated tbr 1 hr at room temperature in the appropriate naphthyl substrate dissolved in buffer together with 50 mg coupling compound, Fast Blue B salt (Sigma). Caseinolytic activity was localized by incubating the agarose gel after electrophoresis in 1% casein (Ham-

635

merstein, BDH, Poole, U.K.) in 0.2 M Tris-HC1 buffer, pH 7.8, for 30 min at 37°C. The gel plate was then rinsed with distilled water and incubated in a humid chamber for another 15 min at 37°C, rinsed again with water, fixed in 2% acetic acid and dried before protein-staining with Coomasie Brilliant Blue. Elastinolytic activity was detected by covering the gel plate after electrophoresis with a second 1% agarose gel, containing 0.25% elastin congo red (Sigma) cast on a plastic support (Gel-Bond). After incubation of the gel sandwich for 4 hr at 37°C, the elastin°containing gel was removed and covered with filter papers and absorbent papers and pressed (1 kg) to transfer digested elastin congo red to the filter paper. Amylase activity was detected in a similar manner by covering the electrophoretic plate with a I% agarose gel containing 0.4% blue starch polymer (1 tablet/50 ml, Phadebas Amylase test, Pharmacia, Uppsala, Sweden) in 0.02 M phosphate buffer, pH 7.8 + 0.05 M NaCI for 30 min (Ceska, 1971). The starch-containing gel was then treated as described above for the elastin gel. Gel filtration Freeze-dried pancreas juice was dissolved, 30 mg/ml, in 0.05 M Tris-HCl buffer + 0.15 M NaC1, pH 7.5, and activated with 0.3 mg enteropeptidase/ml for 30 min at 37°C. 0.2ml juice was injected on a gel filtration column (1 x 30 cm), Superose TM 12 (Pharmacia, Uppsala, Sweden), equilibrated with buffer and connected to a HPLC-system (Varian 5000, Waters Ass. Inc., Milford, Mass., U.S.A.). The flow rate was 0.5 ml/min and l min fractions were collected, which were electrophoresed and analyzed for enzyme activities or stained for protein.

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Fig. 1. Schematical drawing of the enzyme activities obtained with activated porcine pancreas homogenate (la) and juice (lb) after electrophoretical separation in agarose gel, at pH 8.6, and incubation with the various substrates indicated. The hatched bands indicate weak activity, while filled bands indicate strong activity. The electrophoretical migration in cm (approx. mid. point) from the application site (arrow) is indicated either in the anodal ( + ) or cathodal ( - ) direction and a protein-stained electropherogram (photo) is given as reference.

BERTIL G . OHLSSON et al.

636 RESULTS

The summarized patterns of the enzyme activity bands obtained after agarose gel electrophoresis with the different substrates are shown for activated porcine pancreas homogenate in Fig. 1a and for the juice in Fig. lb. For each substrate, the incubation time and the enzyme concentration were chosen to find the optimal pattern with distinct and well separated bands. Pancreas homogenate and the commercial preparation, pancreatin, showed almost identical patterns with the substrates studied. Elastinolytic activity was found in two bands in positions ( - 0 . 7 ) and ( - 1.5) in both the homogenate and the juice. With CBZ-AIa-flNE, Suc-(Ala)3-pNA and MeO-Suc-Ala-Ala-Pro--Val-pNA, all of which are synthetic elastase substrates, identical results were obtained, with bands in positions ( + 2,4) and ( - 1.4). With CBZ-AIa-/3NE three additional weak bands ( + 3.5, +0, - 2 . 5 ) were obtained in the homogenate and two bands (+3.5, +2.9) in the juice. Using S u c - A l a - A l a - P r o - P h e - p N A , Bz-Tyr-pNA and Suc-Phe-pNA as chymotrypsin substrates three bands were obtained (+3.4, +0, - 0 . 5 ) although the bands at ( + 3.4) and ( + 0) were weak in most cases with the two latter substrates. With S u c - A l a - A l a - P r o - P h e - p N A additional bands at (+2.4) and (+0.6) were obtained. With Bz-TyrpNA one extra band at (+2.7) appeared in the juice. With the trypsin-chymotrypsin substrate Ac-Phe-flNE, bands were obtained in positions corresponding to the bands obtained with the chymo-

trypsin substrates as evaluated together ( + 3.4, + 2.4, +0.6, + 0 and -0.5), with the exception of an extra cathodal band ( - 2 . 4 ) . Furthermore, three bands in positions ( + 1.7, +0.8 and - 1 . 6 ) were obtained in the homogenate. Using the trypsin substrate, Bz-Arg-pNA, one band at ( + 1.7) was found in the homogenate. In both the homogenate and the juice a band at ( - 2 . 4 ) was identified at the same position as with Ac-Phe-/~NE. Caseinolytic activity was found corresponding to all bands obtained with these substrates, except for the Ac-Phe-//NE band at (+0.8) obtained with the homogenate. Using CN-Phe as carboxypeptidase A substrate, one band (+2.0) was identified. With blue starch polymer as amylase substrate, one zone, probably containing two bands immediately anodal to the application site (+0.5), was found. Figure 2 shows the chromatogram obtained after gel filtration of activated pancreas juice and the trypsin-chymotrypsin activity bands obtained in the fractions after agarose gel electrophoresis and incubation with Ac-Phe-flNE. As judged from the elution of maximal enzyme activity the retention times were: the band in position +3.4 at 28.0 min, three bands (+0.8, +0.6, +0) at 28.2, 28.5 and 28.7 min respectively, two bands ( - 1 . 7 , - 2 . 4 ) at 30 min and two bands ( + 0, - 0 . 5 ) at 31.8 min. Using the elastase substrate, Suc-(Ala)3-pNA, two bands ( + 2.4, - 1.4) were found with elution times of 31.0 and 31.5 min respectively, and with CN-Phe, carboxypeptidase A activity (+2.4) was eluted at 28.5rain. Amylase

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Fig. 2. Gel filtration (HPLC) of activated pancreas juice on a SuperoseTM 12 column (1.0 × 30cm) equilibrated in 0.05 M Tris-HC1 buffer+ 0.15 M NaCl pH 7.5. The flow rate was 0.5 ml/min and one minute fractions were collected. The protein-containing fractions were analyzed for trypsin-chymotrypsin activity after agarose gel electrophoresis with the substrate Ac-Phe-flNE.

Pig pancreatic hydrolases

637

Table 2. Summaryof propertiesof the porcinepancreatichydrolasesidentifiedin homogenateand juice; migration distance in cm after agaros¢gel elcctrophoresisin the anodal (+) and cathodal ( - ) directions, enzyme activitiesobtainedwith the varioussubstrates(+ indicatesweak activityand + + indicatestrong activity), and approximateretention times after high performance gel filtration on Superos¢TM 12 SUBSTRATES

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activity was eluted in several fractions, between 27 and 33 min, indicating a non-exclusion interaction with the gel matrix. DISCUSSION The identification and characterization of the porcine pancreatic enzyme activity bands obtained after electrophoretical separation were primarily based on their activities towards the various substrates used, their electrophoretical migration and their retention properties on gel filtration. These characteristics were compared to those obtained in other studies on purified hydrolases and summarized in Table 2. The results obtained with pancreas homogenate, juice and pancreatin appeared almost identical indicating that no apparent differences exist between the enzymes when stored in zymogen granulae in the gland or when secreted in pancreas juice. Of the two elastinolytic bands found, the more cathodal one was localized in a similar position ( - 1 . 5 ) as the bands obtained with the synthetic elastase substrates Suc-(Ala)3-pNA and MeO--SucA l a - A l a - P r o - V a l - p N A . These activities presumably represent elastase I, which when purified has been shown to solubilize elastin and hydrolyze Ac-(AIa)3-pNA (Gertler et al., 1977). The second elastinolytic band ( - 0 . 7 ) was localized at a similar position as the bands found with the 4 chymotrypsin B.C. 19/7--D

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substrates. The enzyme was identified as elastase II (also called elastase B) which can solubilize elastin and is highly active against typical chymotrypsin substrates (Gertler et al., 1977; Ardelt, 1974; Vered et al., 1985). Thus, though both elastase I and II are active for elastin, elastase II principally cleaves peptide bonds were the carboxyl group is contributed to be hydrophobic amino acids, while elastase I mainly cleaves at alanine. The third elastase-like proteinase with anodal (+2.4) activity to the synthetic elastase substrates, but showing no elastinolytic activity, was identified as protease E (Kobayashi et al., 1978). Three major bands, probably representing the three chymotrypsins A, B, C were obtained when evaluating the results with the chymotrypsin substrates together. The most anodal one ( + 3.4) which was eluted earliest u p o n gel filtration represents chymotrypsin C, which has the highest described molecular weight, 29,000 dalton, and is the most anionic o f t b e chymotrypsins (Desnuelle et al., 1970). The chymotryptic band (+0.6) localized electrophoretically in between the two others was eluted second on gel filtration (at 28.5 rain) and represents chymotrypsin B, with a reported molecular weight of 26,000 dalton (Gratecos et al., 1969). The third chymotryptic band (+0), was eluted slightly after chymotrypsin B upon gel filtration (at 28.7 min). This band represents chymotrypsin A, the most cationic

638

BERTIL G. OHLSSON et al.

form and with the lowest molecular weight, 24,600 enzymes detected. Bz-Tyr-pNA and Suc-Phe-pNA dalton, reported (Desnuelle et al., 1970). The three are considered to be specific for the chymotrypsins chymotrypsins showed similar but not identical activ- (Bundy, 1962; Nagel et al., 1965) and are commonly ities to the 4 substrates tested, indicating differences used (Yen et al., 1977). However, in this study these in their respective enzyme specificities. Thus, both substrates and especially Suc-Phe-pNA, appears to chymotrypsin A, B and C showed activity to be more "specific" for elastase II than for the chymoAc-Phe-flNE and S u c - A l a - A l a - P r o - P h e - p N A , trypsins. The method used in this study, electrophoretical while chymotrypsin B showed no activity to Bz-Tyr-pNA and Suc-Phe-pNA and chymotrypsin separation of pancreatic hydrolases followed by detection by enzymoblotting, appears to be a useful C showed only weak activity to these. The isoenzyme activity found in two bands approach for investigating enzymes and their various ( + 1.7 and - 2 . 4 ) in the pancreas homogenate with forms in complex solutions. In addition, the method the substrate Bz-Arg-pNA, represents anodal (Voy- offers advantages when investigating specificity and tek and Gjessing, 1971) and cathodal trypsin (Travis sensitivity of synthetic substrates. and Liener, 1965). Although not found here in the SUMMARY activated pancreatic juice, anodal trypsin can be found when the electrophoresed zymogen in the juice A survey of the porcine pancreatic hydrolases was was investigated by enzymoblotting (Ohlsson et al., performed in activated pancreas juice and homoge1986) and anodal trypsin appears to be particularly nate after electrophoretic separation in agarose gel, at unstable in activated probes. pH 8.6. Eight proteinases plus carboxypeptidase A Carboxypeptidase A activity was obtained as an and amylase were identified using substrates of varianodal band (+2.4) with CN-Phe. This is in agree- ous specificity, either directly in the gel or by enment with results obtained by Martinez et al. (1981) zymoblotting. and Folk and Schirmer (1963) using the substrate Three elastase-like proteinases were characterized: hippuryl-L-phenylalanine for identification. elastase I with activity for elastin and the synthetic Amylase activity was localized in a zone (+0.5), elastase substrates S u c - A l a - A l a - A l a - p N A and probably containing two close bands, representing M e O - S u c - A l a - A l a - P r o - V a l - p N A ; protease E with the two amylases isolated from porcine pancreas activity only for the synthetic elastase substrates and homogenate (Cozzone et al., 1970). elastase II with activity for elastin as well as activity It should be pointed out that the bands obtained for the typical chymotrypsin substrates after electrophoresis demonstrate the localization of S u c - A l a - A l a - P r o - P h e - p N A , Bz-Tyr-pNA and the active forms of the enzymes, which are not always Suc-Phe-pNA. Chymotrypsin A, B and C showed identical with the localization of the corresponding similar but not identical, activities for the chymozymogen forms (Ohlsson et al., 1986). trypsin substrates mentioned above and When choosing substrates to determine specific Ac-Phe-flNE and were identified merely by their enzyme activities in solutions containing a mixture of different behavior on gel filtration and electroproteinases the results of this investigation should be phoresis: chymotrypsin C was the most anionic with considered. The ideal substrate should be both the highest apparent molecular weight; chymotrypsin specific and sensitive to a particular enzyme. A was slightly cathionic and had the lowest molecular Ac-Phe-flNE, for example, was found to be a sensi- weight and chymotrypsin B had properties in betive but a non-specific substrate, since both trypsin, tween. Anodal and cathodal trypsin were identified chymotrypsin as well as protease E and elastase II using the specific substrate Bz-Arg-pNA. Finally, showed activity with it. On the other hand, carboxypeptidase A was identified with CN-Phe and B z - A r g - p N A was found to be a specific substrate for amylase with blue starch polymer. both trypsins, although no discrimination between The simple method described, electrophoretical the isoenzymes was possible. separation followed by characterization with specific Increasing the length of the synthetic substates has substrates, allows direct investigation of enzymes in been a way to increase their specificity and sensitivity. complex solutions without purification. In addition, The elastase substrates with 3-4 amino acid residues, the method is advantageous when investigating subSuc-(Ala)3-pNA and M e O - S u c - A l a - A l a - P r o - strate specificity and sensitivity. Val-pNA, were apparently more specific than CBZ-Ala-flNE, the latter showing weak activity also Acknowledgements--The excellent and willing technical asto the trypsins and the chymotrypsins. Finding an sistance of Mrs Inger Mattsson is very much appreciated. ideal substrate for the elastase-like enzymes appears Peptide synthesis in collaboration with Bj6rn Ekberg is very difficult. Elastase I shows activity against both elastin much appreciated. Funds received from the Swedish Counand the amino acid/peptide elastase substrates. Elas- cil for Natural Science Research, the Swedish Council for tase II, besides its elastinolytic activity, shows activity Forestry and Agricultural Research and Director A. against typical chymotrypsin substrates, while P¢thlsson's Foundation supported these studies. protease E shows activity to the synthetic elastase REFERENCES substrates but no elastinolytic activity. On the other hand, these differences, for the present, form a basis Ardelt W. (1974) Partial purification and properties of for discrimination between the elastase enzymes. porcine pancreatic elastase II. Biochim. biophys. Acta 341, Of the chymotrypsin substrates investigated, 318-326. S u c - A l a - A l a - P r o - P h e - p N A , was more sensitive Bieth J., Spiess B. and Wermuth C. G. (1974) The synthesis and analytical use of a highly sensitive and convenient than the shorter substrates, Bz-Tyr-pNA and substrate of elastase. Biochem. Med. 11, 350-357. Suc-Phe-pNA, resulting in a greater number of

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