Protease I Inhibitor System in Fish Muscle: A Comparative Study

Comp. Biochem. Physiol. Vol. 119B, No. 1, pp. 101–105, 1998 Copyright  1998 Elsevier Science Inc. All rights reserved.

ISSN 0305-0491/98/$19.00 PII S0305-0491(97)00282-4

Protease I Inhibitor System in Fish Muscle: A Comparative Study O. Pe´rez Borla,1 C. B. Martone,2 and J. J. Sa´nchez 1,2 1

Departamento de Qui´mica and 2 Instituto de Investigaciones Biolo´gicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Casilla de Correo 1245, 7600 Mar del Plata, Repu´blica Argentina

ABSTRACT. A protease and a trypsin inhibitor were partially purified from skeletal muscle of fishes phylogenetically different. The protease fraction from each fish species studied was immunologically similar to protease I from white croaker. Each endogenous inhibitor fraction inhibited protease I fraction from other fish species. The present results suggest that protease I inhibitor system is present in all fish species studied. comp biochem physiol 119B;1:101–105, 1998.  1998 Elsevier Science Inc. KEY WORDS. Fish, muscle, protease, protease inhibitor, proteinase, proteolysis, serine protease

INTRODUCTION Several non-lysosomal proteolytic activities have been observed in mammalian, avian and, more recently, fish muscle (1,19,20). At present, there are many reports on alkaline proteases in skeletal muscle of several fish species; most of them are thermostable and show high M r (3,8,12,14,21). Also, it has been generally assumed that some changes in functional (i.e., gelation) and organoleptic (i.e., taste) properties of fish muscle are a consequence of proteolytic activity. The alkaline proteases could play an important role in turnover and post-mortem modifications of fish muscle proteins (2,5,12,13,22–25). We reported the presence of an alkaline protease I inhibitor system in white croaker and hake skeletal muscle. Protease I is a trypsin-like serine protease of high M r that shows optimum activity at a lightly alkaline pH. This protease is able to act on structural and contractile proteins, and it may play an important role in both physiological and post-mortem fish muscle proteolysis (3,4,8). Protease I is quite different from the multicatalytic proteinase (MCP) or proteasome, which is also present in fish skeletal muscle in M r (3), subunit composition (9,10), action on myofibrils (3) and sensitivity to specific and endogenous inhibitors (3,8). There exists several enzyme-inhibitor systems, some protease inhibitors whose target enzyme is as yet unknown, and several extralysosomal proteolytic systems (MCP, calpains) whose distribution is ubiquitous (1,19,20). We have speculated that the protease I inhibitor system previously reAddress reprint requests to: O. Pe´rez Borla, Instituto de Investigaciones Biolo´gicas (FCEN -UNMdP), CC 1245, (7600) Mar del Plata, Argentina. Fax 54-23-753150; E-mail:[email protected] Received 15 August 1996; revised 15 July 1997; accepted 23 July 1997.

ported in croaker (3,8) and hake (16) skeletal muscle could also be widely distributed. We report on an initial investigation of the presence of the protease I inhibitor system in skeletal muscle of different fish species. MATERIALS AND METHODS Sources For our work, we chose fish phylogenetically different, caught by commercial ships from the southwest Atlantic Ocean (Argentinian platform—from 36° to 53° S). These fish, valuable commercially as food, were white croaker (Micropogonias furnieri), hake (Merluccius hubbsi), Argentine anchovy (Engraulis anchoita), castan˜eta (Cheilodactylus bergi), rough sead (Trauchurus lanthanis) and sea trout (Cynoscion striatus). The fish were stored on ice (24–48 hr) or frozen (no more than 1 month). Chemicals DEAE-Sephacel and Superose 12 were purchased from Pharmacia Fine Chemicals. Proteinase inhibitors, trypsin and N-blocked peptide 4-methyl-7-coumarine substrate were from Sigma Co. Antibodies were kindly provided by Dr. L. Busconi and Dr. E. Folco. All other chemicals were of highest purity available. Purification Steps Partially purified protease and inhibitor were extracted as previously reported (16). Briefly, muscle (50 g) was homogenized in 2% KCl, chromatographed in DEAE-Sephacel (volume fraction, 1 ml) equilibrated with 50 mM NaCl–5

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TABLE 1. Biological classification of fishes studied

Class Subclass Infraclass Super order Order Suborder Family Genus Species

Osteichthyes Actinopterygii Teleostei Paracantopterygii Gadiforme Gadoidei Merluciidae Merluccius hubbsi Hake

Clupeomorpha Clupeiformes Clupeoidei Engraulidae Engraulis anchoita Argentine anchovy

Acanthopterygii Perciformes Percoidei Scianidae Carangidae Micropogonias Cynoscion Trauchurus furnieri striatus lanthanis White Sea Rough croaker trout sead

Cheilodactylidae Cheilodactylus bergi Castan˜eta

This classification is according to Menni et al. (17).

mM borate buffer, pH 7.5, and eluted with 50–300 mM NaCl in the same buffer. We performed another purification step: the active pooled fractions from DEAE-Sephacel chromatography were concentrated and chromatographied in Superose 12 (HR 10–30, FPLC) in 50 mM NaCl– 20 mM Tris–HCl buffer (volume fraction, 1 ml). Typically, DEAE-Sephacel and Superose 12 pools contained 1–2% and 0.2–0.3%, respectively, of the protein present in crude extract. All concentration steps were performed using Centricon10 concentrator (Amicon). Protein concentration was determined by the method of Lowry et al. (15) using bovine seroalbumin as standard.

Assays of Proteolytic and Inhibitory Activities For DEAE-Sephacel fractions and for pools, the proteolytic activity was determined at 37°C in 100 mM Tris–HCl buffer, pH 8.5, using azocasein as substrate; the reaction was stopped after 3 hr with trichloroacetic acid (final concentration 5% w/v). One enzyme unit is defined as the amount of enzyme that produce a ∆A335 per hour 5 1, under the assay conditions. For Superose 12 fractions, the proteolytic activity was determined at the same pH and temperature conditions but using N-t-Boc-Val-Pro-Arg 7-amino-4methylcoumarin (Boc-Val-Pro-Arg-NH-Mec) as substrate; the reaction was stopped after 90 min with soybean trypsin inhibitor (final concentration, 1 mg/ml). Fluorescence of liberated 7-amino-4-methylcoumarin was measured using a primary filter of 370 nm (excitation) and a secondary filter of 470 nm (emission). The activity was expressed as an increase of fluorescence and given in arbitrary units. Specific inhibitory assays were performed similarly after preincubating the enzyme fractions with each inhibitor 30 min at room temperature. Endogenous inhibitory activity was assayed, at 37°C, on trypsin (1 unit) using azocasein as substrate in 100 mM Tris–HCl buffer, pH 8.0, and stopped, after 40 min, with trichloroacetic acid (final concentration, 5% w/v). One inhibitor unit is defined as the amount of inhibitor fraction

that produce 50% inhibition of trypsin under the assay conditions. Analytical Procedures Electrophoresis analysis under non-denaturing conditions was performed at 4°C on 7.5% polyacrylamide gels, according to Davis (7). For detection of protease activity, 0.2% (w/v) gelatin was copolimerized into the gel. After electrophoresis, the gel was washed in 100 mM Tris–HCl buffer, pH 8.5, and incubated overnight at 37°C in the same buffer. Proteolytic activity was visualized as unstained regions. Staining was performed with Coomassie Brillant blue R250 in 40% methanol, 10% acetic acid or with AgNO3 according to Merril et al. (18). For Western immunoblotting assay, electrophoresed samples were transferred to nitrocellulose membrane. The membrane was blocked with 1% bovine seroalbumine, 0.3% Tween 20, 0.05% NaN 3 in 100 mM Tris–HCl buffer, pH 8.0, and then incubated with rabbit anti- protease I antibody from white croaker. After washing with blocking buffer and incubating with phosphataseconjugated anti-rabbit IgG, immunoreactive bands were revealed using a phosphatase color development reagent. Every assay included white croaker sample with similar purification grade as positive control. Each sample included at least four fish, and we set the same sampling and treatment condition to all fish samples. Similar results were obtained with frozen or chilled samples. RESULTS We analyzed fish of several species to investigate the existence of a protease I inhibitor system such as in white croaker muscle (3,8) Table 1 shows the biological order of the fish species studied. Purification When the dialyzed extracts from white skeletal muscle of all fish species studied were subjected to ion exchange chro-

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matography on DEAE-Sephacel, a single peak of azocaseinolytic activity at 37°C and pH 8.5 was eluted with a salt concentration between 150 and 200 mM NaCl similarly to protease I from white croaker Figure 1A. This activity could be fully separated from a peak of proteolytic activity eluting at higher salt concentration 250–350 mM corresponding to multicatalytic proteinase in white croaker (3,8,9). In all cases, a peak of trypsin inhibitor activity eluting at a salt concentration between 180 and 230 mM was partially separated from the former azocaseinolytic activity (Fig. 1A). In these assays, higher salt concentrations were needed for castan˜eta samples and lower ones for rough sead. From different fish species, specific activities between 1 and 7 U/mg were obtained after pooling azocaseinolytic active ion exchange chromatography fractions. The concentrated pools from DEAE-Sephacel chromatography applied to a Superose 12 (HR 10/30) column showed similar patterns from each fish sample with a unique zone of protease activity eluting at similar volumes (Fig. 1B). The range of specific activity for gel filtration pools was 750–3500 U/mg.

Characterization FIG. 1. (A) DEAE-Sephacel chromatography of rough sead

muscle extract. Proteolytic (r) and trypsin inhibitory (e) activities were determined as described in text, with 100 and 50 ml of column fractions as source of enzyme and inhibitor, respectively. Neither proteolytic nor trypsin inhibitor activities were detected in fractions containing protein not bound to the resin. ——, NaCl gradient (0.050–0.300 M). (B) Superose 12 chromatography of protease pool from DEAESephacel chromatography. Proteolytic activity (r) was assayed as described (see text) with 10 ml of column fraction as source of enzyme using Boc-Val-Pro-Arg-NH-Mec as substrate. . . . , A280 nm. Fractions with proteolytic activity greater than 50% of maximum were pooled as protease and fractions with inhibitory activity greater than 50% were pooled as endogenous inhibitor. Patterns from the other fish samples are close similar.

As shown in Table 2 the serine protease inhibitors tested, aprotinin and SBTI, reduced enzyme activity from all species more drastically than the specific inhibitors of other protease groups. These results suggest the presence of a serine-type protease. Each protease I fraction has been partially inhibited by any endogenous inhibitor studied Table 3. This partial effect of endogenous inhibitor and the partial inhibitory effects of EDTA and pepstatin on some samples may be because protease I and inhibitor samples are not purified yet to homogeneity. Protease I fractions from different fish species showed complex protein electrophoretic patterns Figure 2a. Zymogram of some samples showed more than one gelatinolytic band (Fig. 2b). Despite this fact, the rabbit anti-protease I antibody from white croaker only recognized a single band corresponding in mobility to a gelatinolytic activity one (Fig. 2c).

TABLE 2. Effect of specific protease inhibitors on protease I activity (inhibition %)

Protease source White croaker Hake Rough sead Castan˜eta Sea trout Argentine anchovy

Aprotinin 0.34 U/ml

Soybean inhibitor 0.1 mg/ml

EDTA 1mM

Pepstatin 10 m M

E 64 c 30 m M

50 59 67 68 75 95

50 67 50 89 78 78

0 4 23 0 4 12

20 4 40 45 25 11

10 1 0 0 5 0

The enzyme (final concentration 0.5 U/ml) was preincubated with the respective inhibitor 30 min at room temperature in the assay mixture before addition of Boc-Val-Pro-Arg-NH-Mec solution. Proteolytic activity was determined as described (see text). Data are mean values from three experiments and showed a variation not greater than 5%.

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TABLE 3. Effect of endogenous inhibitor on protease I activity (inhibition %)

Endogenous inhibitor source Protease source White croaker Hake Rough sead Sea trout Castan˜eta

Rough sead

Castan˜eta

Sea trout

A. anchovy

80 40 85 100 20

100 55 70 90 35

30 85 75 — 25

45 90 100 — 35

Every enzyme fraction (final concentration 0.5 U/ml) was preincubated in the presence of each inhibitor fraction (0.75 U/ml) 20 min at room temperature in the assay mixture before addition of azocasein solution. Asocaseinolytic activity was determined as described (see text). Proteolytic activity of inhibitor fractions was negligible. Data are mean values from three experiments and showed a variation not greater than 5%.

DISCUSSION The aim of the present work was to study the protease I inhibitor system in different fish species. We were able to partial purified a protease and a trypsin inhibitor from skeletal muscle of different fish species performing the same methodology. The properties of the protease and the endogenous trypsin inhibitor from each studied species were similar in some respects; our results indicate that in skeletal muscle of rough sead (T. lanthanis), sea trout (C. striatus), castan˜eta (Ch. bergi), Argentine anchovy (E. anchoita), they eluted from DEAE-Sephacel in the same ranges of NaCl concentration. All the samples, including white croaker and hake, eluted at similar volumes in gel filtration chromatography in Superose 12. In the presence of endogenous inhibitors, the proteases extracted from the different fish species showed a similar behavior than white croaker and hake protease I (3,8,16). The observed greater effect of serine-type protease specific inhibitors on protease fractions indicated the presence of a serine-type protease. The partial action of pepstatin and/or EDTA on some samples and the presence of several gelatinolytic bands may indicate the presence of another protease(s); nevertheless, it is clear that protease I exists

in each fish species studied because the specific antibody recognized a unique band among the different proteins present in the protease I fractions. These results and the reciprocal interactions of proteases with endogenous inhibitors support our assumption that a similar protease I inhibitor system is present in fish skeletal muscle despite phylogenetic differences. It seems possible that the protease I inhibitor system has an ubiquitous distribution. At present, we are investigating for its presence in mammal muscle. The present work and further studies could significantly improve the understanding of this protease I inhibitor system from fish muscle to obtain information about its physiological role (4–6,16) and the potential control of its influence on the quality of fish products (5,6,11–13). We thank Dr. L. Busconi and Dr. E.J.E. Folco for antibodies and the Centro de Investigaciones y Tecnologi´a Pesquera for purchased fish and where preparative experiments were done. The authors are affiliated with the Consejo Nacional de Investigaciones Cienti´ficas y Te´cnicas (Argentina). This work was supported in part by the Consejo Nacional de Investigaciones Cienti´ficas y Te´cnicas, the Comisio´n de Investigaciones Cientificas de la Pcia, Buenos Aires and the Universidad Nacional de Mar del Plata (Argentina).

FIG. 2. Electrophoretic and immunologic analysis of protease I fractions from skeletal muscle of different fishes. Samples were electrophoresed in 7.5% polyacrylamide gels under nondenaturing conditions (experimental details in text): (a) protein pattern; (b) activity pattern (gelatin containing gel); (c) Western blot assay. Cr, white croaker; Hk, hake; Rs, rough sead; Ca, castan˜eta; St, sea trout; Aa, Argentine anchovy. The arrows indicate the position of protease I band.

Protease I Inhibitor System

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