IDENTIFICATION OF AN "ALCOHOL DEHYDROGENASE-ACTIVATING" PROTEASE IN GRASS CARP HEPATOPANCREAS AS A CHYMOTRYPSIN

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1 Vol. 43, No. 6, December 1997 BIOCHEMISTRY end MOLECULAR BIOLOGY INTERNATIONAL Pages '1239 IDENTIFICATION OF AN "ALCOHOL DEHYDROGENASE-ACTIVATING" PROTEASE IN GRASS CARP HEPATOPANCREAS AS A CHYMOTRYPSIN King-kwan Lau, Elaine Yee-man Chan and Wing-ping Fong* Received September 9, 1997 Received after revision October 20, 1997 Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong SUMMARY: Previous investigation [Tsui et al. (1996) Biochim. Biophys. Acta 1269: 41-46] showed that two active forms of alcohol dehydrogenase can be purified from grass carp. The use of a protease inhibitor and the results of SDS-PAGE analysis of the enzymes suggest that one form (ADH-C) is a proteolytic product of the other (ADH-I). In this study, the protease responsible for the cleavage was purified. The cleavage enzyme had a subunit molecular weight of 28 kda. An inhibitor study identified it as a serine protease. It exhibited a strong chymotrypsin activity in both esterase and amidase assays with a ph optimum in the range The purified chymotrypsin also cleaved the intact grass carp ADH-I into the two-fragment ADH-C, with an accompanying increase in enzyme activity. A similar effect was not found using horse liver alcohol dehydrogenase. Key words: activation, alcohol dehydrogenase, chymotrypsin, grass carp, proteolysis INTRODUCTION During enzyme purification, it is of paramount importance to prevent unwanted proteolysis. It is necessary to ensure that the protein obtained represents the physiological form and does not arise as a consequence of sample material processing and analysis. The most common way to follow enzyme purification is by monitoring changes in its activity. In cases where the proteolytic product is not active, proteolysis will only lower the yield of the Abbreviations: ADH, alcohol dehydrogenase; BAEE, benzoyl-arginine ethyl ester; BTEE, benzoyl-tyrosine ethyl ester; 3,4-DCI, 3,4-dichloroisocoumarin; E-64, L-transepoxysuccinyl-leucylamide-(4-guanidino)-butane; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethanesulfonyl fluoride; SBTI, soybean trypsin inhibitor; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Suc-AAPF-NA, succinyl-l-ala- L-Ala-Pro-L-Phe-p-nitroanilide; TPCK, tosyl-l-phenylalanine chloromethyl ketone. *Corresponding author. Fax: (852) ; b348738@mailserv.cuhk.edu.hk /97/ ) /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 purification as the cleavage product will be removed during the process. However, if the proteolytic product also possesses activity, the situation is more complicated, such is the case in grass carp (Ctenopharygodon idellus) alcohol dehydrogenase (ADH)(I). ADH (E.C.I.I.I.1) are oxidoreductases catalyzing the oxidation of alcohols to the corresponding aldehydes/ketones with the reduction of NAD to NADH. The enzyme is common in nature across a wide range of living organisms. The investigation has been made in order to attempt to understand aspects of the evolution of ADH from a number of lower vertebrates. For example, in fish, ADH has been isolated and characterized from cod (2,3), rainbow trout (4,5), pickerel (6) and grass carp (L7). In a previous investigation of grass carp ADH, two different forms of enzyme were obtained (1). In the presence of benzamidine and dithiothreitol, the ADH (named as ADH-I) had a subunit molecular weight of 40 kda, similar to that of other median-chain length ADH (8). However, in the absence of these two compounds (inhibitors), the ADH (named ADH- C) obtained consisted of two different types of subunits, namely, 27 and 13 kda. These results, together with other evidence presented therein, led to the suggestion that ADH-C is a proteolytic product of ADH-I. It is of note that after the specific cleavage, both the Km and the Vmax values of the enzyme were increased. This is the first example of a catalytically active ADH cleavage product. Thus, the proteolyic cleavage, although it arose from an artefact during purification, may be investigated in order to attempt to understand aspects of the structure-function relationship of the enzyme. In this investigation, the protease responsible for the proteolytic activation has been purified. Based on its susceptibility towards protease inhibitors and its substrate specificity, the "ADH-activating protease" was identified as a chymotrypsin. While the purified chymotrypsin could activate grass carp ADH, it failed to have any significant effect on the ADH of horse liver. METHODS Purification of "'ADH-activating" protease. Grass carp were obtained from a local market and were killed by a blow on the head. The hepatopancreas was removed and frozen at -70~ before use. In a typical preparation, 20 g of hepatopancreas were homogenized in 40 ml of 10 mm Tris-HC1, ph 7.9. The homogenate was centrifuged at 47,500 g for 1 h. The supernatant fluid was heated at 60~ (twice, 5 min each), and centrifuged again at 47,500 g for 30 min. The final supernatant fraction was dialyzed against 10 mm Tris-HCl, ph 7.9. After dialysis, it was applied to a DEAE-cellulose column (5 x 10 cm) and equilibrated with the dialyzing buffer. The column was eluted with 0.1 M NaCI in the same buffer. The 1232

3 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL protein peaks were pooled and the one exhibiting "ADH-activating" activity (vide infra) was concentrated by ultrafiltration. The sample was dialyzed against 10 mm Tris-HCl, ph 7.5 before being loaded onto a Q-Sepharose column (1.5 x 12 cm). The column was eluted with a linear gradient of M NaCI in the dialyzing buffer. The protein peaks were pooled and the active one was concentrated. After dialysis against 10 mm sodium phosphate, ph 7.5, the protease preparation was loaded onto a benzarnidine-sepharose column (1 x 10 cm) and washed with the same buffer. The active fractions were pooled, concentrated and the preparation was ready for analysis. "ADH-activating" assay. The intact grass carp ADH-I, isolated as described in (1), was used as the substrate for the assay. The protease preparation was incubated with ADH-I in 10 mm sodium phosphate, ph 7.5 at 30~ Aliquots were removed at intervals for ADH activity assay. ADH activity was determined at 25~ by spectrophotometrically following the formation of NADH at 340 nm (~340 = 6220 M-lcm 1) in an assay medium containing 2.5 mm NAD +, 0.1 M glycine-naoh, ph 10.0, and 40 or 250 mm ethanol as substrate. Caseinolytic assay. The assay was carried out with 1% casein in 0.1 M Tris-HC1, ph 8.0 containing 10 mm CaC12. After incubation at 37~ for 15 rain, the reaction was terminated by adding the same volume of 5% trichloroacetic acid. The amount of acid soluble product was determined by the method of (9). Esterase assay. The esterase assay was performed at 25~ with 0.5 mm BTEE, 10 mm CaC12, 5% methanol in 0.1 M Tris-HC1, ph 8.0. The reaction was followed by the increase in absorbance at 256 nm (e256 = 964 M-lcm-1). One unit of esterase activity refers to the hydrolysis of 1 Ixmol BTEE per rain. Amidase assay. The amidase assay was performed at 25~ with 0.3 mm Suc-AAPF- NA, 10 mm CaC12 in 0.1 M Tris-HCl, ph 8.0. The reaction was followed by the release of p-. nitroanilide and the increase in absorbance at 410 nm (e4a0 = 8480 Mlcml). One unit of amidase activity refers to the production of 1 lamol p-nitroanilide per rain. SDS-PAGE & isoelectric focusing. SDS-PAGE was carried out according to the method of (10). PMSF (2 mm) was included in the sample buffer to minimize proteolysis. The gels were stained with Coomassie Blue. Isoelectric focusing was performed on the PhastSystem (Pharmacia) IEF gel and stained according to the manufacturer's recommendation. Protein determination. All protein determinations were performed by the method of (9), using bovine serum albumin as the standard. RESULTS The "ADH-activating" protease was purified from grass carp hepatopancreas by monitoring its activating effect on grass carp intact ADH-I. Chromatography of the crude extract on DEAE-cellulose separated the proteins into two fractions, the breakthrough D-I and the adsorbed D-II (Fig. 1A). The D-II fraction contained the "ADH-activating" activity. Removal of the contaminating proteins was achieved by another, more efficient anion exchanger Q-Sepharose which was chromatographically developed with a linear gradient of M NaC1. Four major peaks were resolved (Fig.lB). Among them, Q-Ill, i.e. the one eluted with ~0.12 M NaCI, possessed the "ADH-activating" activity. The final purification 1233

4 2.o I A o-,, I, o-,,~ ~.ol /',.o.,~+o, < o.o j+ ' i ~,. 0"3' B Q-II O-Ill 9 Q-IV (.) eq 0.1 ~ 0.1 < m 0.0 w - w 0.0 z ~ 0.4 e~ 0.2 < o.+fc, Fraction Number Figure 1: Purification of "ADH-activating" protease. (A) Chromatography of the supernatant fraction, after heat treatment, on DEAE-cellulose, The column (5 x 10 cm) was washed with 10 mm Tris-HCl, ph 7.9 and eluted with 0.1 M NaC1 in the same buffer. Fractions of 8 ml were collected. 03) Chromatography of D-II on Q-Sepharose. The column (1.5 x 12 cm) was eluted with a linear gradient of M NaC1 in 10 mm Tris-HC1, ph 7.5. Fractions of 5 ml were collected. (C) Chromatography of Q-Ill on benzamidine-sepharose. The column (1 x 10 cm) was washed with 10 mm sodium phosphate, ph 7.5. Fractions of 2 ml were collected. was achieved by the benzamidine-sepharose column which retarded the "ADH-activating" protease (Fig.lC). The final preparation obtained 03-I) was homogeneous by SDS-PAGE analysis. A single band at 28 kda was observed (Fig.2). An isoelectric focusing experiment also showed a single band with an isoelectric point of 5.8 (data not shown). The effects of a number of protease inhibitors, including reversible and irreversible ones, were tested on the activity of the purified protease (Table 1). Among them, SBTI, PMSF, TPCK and 3,4-DCI significantly inhibited the activity of the protease; whereas leupeptin, pepstatin A, EDTA, 1,10-phenanthroline, aprotinin, benzamidine, iodoacetic acid and E-64 had only minor effects, if any, on the enzyme. The purified protease did not exhibit any activity with the trypsin-specific ester substrate BAEE (data not shown). It was however most reactive with the chymotrypsin-specific ester substrate BTEE, where the 1234

5 kda o "'-" 2o.1 Figure 2: SDS-PAGE of samples at different stages of purification. Lane 1: crude extract; lane 2: supernatant fraction obtained after heat treatment; lane 3: Fraction D-II; lane 4: Fraction Q-Ill; lane 5, Fraction B-I; lane 6: molecular weight markers (Pharmacia) Table 1: Effects of some inhibitors on "ADH-activating" protease activity Inhibitor Concentration Residual Activity (%) caseinolytic esterase amidase (A) Reversible Inhibitors: Leupeptin 0.1 mg/ml Pepstatin A 0.1 mg/ml 87.0 ND a ND EDTA 10 mm ,10-Phenanthroline 10 mm 89.7 ND 96.0 Aprotinin 0, l mg/ml 91.2 ND ND Benzamidine 10 mm 86.9 ND 96.2 SBTI 0.2 mg/ml ) Irreversible Inhibitors~: PMSF 1 mm TPCK 1 mm Iodoacetic acid 10 mm ,4-DCI 0.1 mg/mg E mg/mg and: not determined, as the inhibitor itself has strong absorbance at the wavelength used to follow enzyme activity. bthe irreversible inhibitor was first incubated with the purified protease at 25~ for 30 min before an aliquot was taken for activity assay. specific activity was 102 U/rag. It also exhibited high activity (8.22 U/mg) with the chymotrypsin-specific amide substrate Suc-AAPF-NA. For both of the above activities, the ph optimum was within the range 7.5 to 8'.5 (Fig3). Upon incubation, the purified grass carp protease caused a progressive activation of grass carp ADH-I. The percent activation depended on the concentration of substrate used. With 40 mm ethanol, only a 50% increase in activity was found after 6 h whereas the 1235

6 gl ph t t~ 3; < Figure 3: ph-activity profile of grass carp "ADH-activating" protease. Enzyme activity was determined using BTEE (0) or Suc-AAPF-NA (O) as substrate. The reaction buffer used was sodium acetate (ph 5-6), HEPES-NaOH (ph 6-8), TRIS-HCI (ph 7.5-9) and glycine- NaOH (ph ). All the reaction buffers were 0.1 M and contained 10 mm CaCI2. activation was more than 5.4 fold greater when the ADH activity was measured with 250 mm ethanol (Fig.4A). Such activation was accompanied by the cleavage of the 40 kda subunit to form the 27 and 13 kda subunits (Fig.4B). The proteolytic activation of ADH-I was slower in the presence of the coenzyme. With 250 mm ethanol as substrate, the ADH was activated to approx. 1.8 fold after 1 h of treatment in the presence of 10 mm NAD +, much lower than the 3.2 fold obtained in its absence (Fig.4A). In contrast with the results of the grass carp enzyme, horse liver ADH was resistant to proteolytic activation. The activity of the horse liver enzyme, assayed at both 40 and 250 mm ethanol and the subunit structure, were unchanged under the same proteolytic treatment conditions (data not shown). DISCUSSION The "ADH-activating" protease was purified by a combination of heat and chromatography treatments. In the crude extract, grass carp ADH and the protease were present together. The grass carp ADH may have included both the intact form (ADH-I) and the cleaved, i.e. activated, form (ADH-C); this potential mixture complicates the interpretation of measurements of the "ADH-activating" activity. Thus, it is essential to remove the ADH from the protease preparation at the outset. In a previous investigation (1), an ADH-free crude protease preparation was obtained by passing the sample through DEAEcellulose and Affi-gel Blue agarose. It was successful but the procedure was cumbersome. In the investigations reported here, it was found that a heat treatment at 60~ removed ADH. All the ADH in the crude extract was heat inactivated while at least 75% of the protease 1236

7 6oo BIOCHEMISTRY ond MOLECULAR BIOLOGY INTERNATIONAL B m.~ 450 A 300 w m Incubation Time, min Figure 4: Activation of ADH by the protease. The protease was incubated with ADH at a ratio of 1:20, in the presence (square) and absence (circle) of 10 mm NAD +. (A) Aliquots were removed at intervals and assayed for ADH activity with 40 (closed symbols) or 250 (open symbols) mm ethanol as substrate. 03) SDS-PAGE analysis of the sample after 0 (lane 1) and 6 (lane 2) h incubation. Lane 3: molecular weight markers. activity remained. Consequently, heat treatment enabled the "ADH-activating" assay to be performed under a well-defined conditionl For an initial classification of the protease, the susceptibility of the protease to a group of protease inhibitors was studied. The serine protease inhibitors, including SBTI, PMSF and 3,4-DCI significantly inhibited the caseinolytic activity of the protease. In contrast other protease inhibitors, including those for aspartic (pepstatin A), metallo (EDTA, 1,10- phenanthroline) and cysteine (leupeptin, iodoacetic acid, E-64) proteases had only minor effects on the enzyme. The overall conclusion of the inhibitor studies suggested that the grass carp "ADH-activating" protease was a serine protease. The inhibitor study also provided some clues as to the type of serine protease present. TPCK, which selectively affects chymotrypsin, decreased the activity of grass carp "ADHactivating" protease; whereas benzamidine and leupeptin, well known trypsin-specific inhibitors, failed to lower its activity, Consequently, the purified protease was more likely to be a member of the chymotrypsin type than a trypsin type enzyme. This suggestion is also consistent with the observation that the grass carp protease was resistant to inhibition by aprotinin. Aprotinin is more specific for trypsin than chymotrypsin. It inhibits some but not all of the different forms of chymotrypsin. For example, it is relatively ineffective towards the chymotrypsins from cod (11) and chym,otrypsin II from rainbow trout (12). The substrate specificity of the protease strengthens the proposal that the identity of the "ADH-activating" protease as a chymotrypsin. It was active towards the chymotrypsinspecific substrates BTEE and Suc-AAPF-NA. The inhibition patterns of the esterase and 1237

8 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL amidase activities by the different protease inhibitors resembled that of the caseinolytic activity (Table 1). The ph-activity pattern was also similar for both esterase and amidase activity. Optimal activity was measured in the ph range 7.5 to 8.5, similar to that found for other fish chymotrypsins ( 11-15). The structural properties of grass carp chymotrypsin also resembled those of other fish chymotrypsins. SDS-PAGE analysis indicated an apparent molecular weight of 28 kda, comparable to other fish chymotrypsins with molecular weights ranging from 25 to 29 kda (11,12,14-17). Like the other fish chymotrypsins, the single band pattern was not affected by treatment with 13-mercaptoethanol, suggesting that the grass carp enzyme was composed of a single polypeptide chain. In this regard, the properties of the fish chymotrypsin are in sharp contrast with the bovine chymotrypsin which splits into three polypeptide chains under reducing conditions (18). However, it has to be cautioned that a small peptide chain of the grass carp enzyme may be "lost" on SDS-PAGE, as is the case with the cod chymotrypsin (19,20). Fish chymotrypsins also differ from their bovine counterpart in that they are more acidic (11,12,14,17). The present result is also consistent with this general observation. The grass carp enzyme had an isoelectric point of 5.8, much lower than that of bovine chymotrypsin (p1-8.5) (21). Incubation of the purified grass carp chymotrypsin with intact grass carp ADH-I resulted in a proteolytic activation of the enzyme, the intact ADH subunit being cleaved into two polypeptide chains in the process. Therefore, it is obvious that chymotrypsin was the protease responsible for the cleavage of ADH-I to ADH-C when the purification was carried out in the absence of benzamidine and dithiothreitol (1). Benzamidine did not inhibit grass carp chymotrypsin (Table 1). Therefore it probably acts indirectly by inhibiting trypsin, thus preventing the activation of the zymogen chymotrypsinogen to the active "ADH-activating" chymotrypsin during the purification process. The grass carp ADH is the only reported example of an ADH in which a well-defined proteolytic product was found to be catalytically active, even more active than the intact enzyme. ADH's have been purified from a number of other fish species (2-6). However, in no case was an active proteolytic enzyme reported, even though there were no particular precautions taken to prevent proteolysis during the purification. It is possible that the particular ADH is not susceptible to proteolytic activation. The well characterized horse 1238

9 liver ADH also failed to exhibit any activation by grass carp chymotrypsin (present study) and a number of commercially available proteases (22,23). The structural basis for the different proteolytic behaviors of the multiplicity of ADHs remains to be investigated. REFERENCES 1. Tsui, H.T., Mock, W.Y., Lau, K.K. and Fong, W.P. (1996) Biochim. Biophys. Acta 1296, Danielsson, O., Eklund, H. and J6rnvall, H. (1992) Biochemistry 31, Danielsson, O., Shafqat, J., Estonius, M., EI-Ahmad, M. and J6mvall, H. (1996) Biochemistry 35, Bauermeister, A. and Sargent, J. (1978) Biochem. Soc. Trans. 6, Tsai, C.S., A1-Kassim, L.S., Mitton, K.P., Thompson, L.E., Van Es, C. and White, J.H. (1987) Comp. Biochem. Physiol. 87B, AI-Kassim, L.S. and Tsai, C.S. (1993) Biochem. Cell Biol. 71, Fong, W.P. (1991) Comp. Biochem. Physiol. 98B, J6rnvall, H., Danielsson, O., Hjelmqvist, L., Persson, B. and Shafqat, J. (1995) Adv. Exp. Med. Biol. 372, Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, Laemmli, U.K. and Favre, M. (1973) J. Mol. Biol. 80, Asgeirsson, B. and Bjarnason, J.B. (1991) Comp. Biochem. Physiol. 99B, Kristjfinsson, M.M. and Nielsen, H.H. (1992) Comp. Biochem. Physiol. 101B, Cohen, T., Gertler, A. and Birk, Y. (1981) Comp. Biochem. Physiol. 69B, Raae, A.J. and Walther, B.T. (1989) Comp. Biochem. Physiol. 93B, Heu, M.S., Kim, H.R. and Pyeun, J.H. (1995) Comp. Biochem. Physiol. l12b, Cohen, T., Gertler, A. and Birk, Y. (1981) Comp. Biochem. Physiol. 69B, Murakami, K. and Noda, M. (1981) Biochim. Biophys. Acta 658, Blow, D.M. (1971) in The Enzymes (Boyer, P.D., ed.), vol. 3, pp , Academic Press, New York. 19. Raae, A.J., Flengsrud R. and Sletten, K. (1995) Comp. Biochem. Physiol. l12b, Leth-Larsen, R., Asgeirsson, B., Th6r61fsson, M., Norregaard-Madsen, M. and Hojrup, P. (1996) Biochim. Biophys. Acta 1297, Laskowski, M. (1955) Methods Enzymol. 2, Rouimi, P., Loomes, K. and J6rnvall, H. (1993) Eur. J. Biochem. 213, Lau, K.K. and Fong, W.P. (1996)Biochem. Mol. Biol. Int. 40,