Myosin heavy chain-degrading proteinase from spear squid muscle

Food Research International,Vol. 28, No. 1, pp. 31-36, 1995

Elsevier Science Ltd Copyright 0 1995Canadian Institute of Food Science and Technology Printed in Great Britain. All rights reserved 0963-9969/95 $9.50 + .OO

ELSEVIER

Myosin heavy chain-degrading proteinase from spear squid muscle Hiizu Ebina,* Yuji Nagasbima,z Shoichiro Ishizaki & Takeshi Taguchi Department of Food Science and Technology, Tokyo University of Fisheries, Minato, Konan, Tokyo 108, Japan

Trypsin-like proteinase responsible for textural deterioration of thermally induced gel was purified to apparent homogeneity from spear squid Loligo bleekeri mantle muscle by chromatography on Sephacryl S-400 and DEAE Sepharose. The enzyme gave a protein band with a molecular weight of 42 kDa on SDS-PAGE. The proteinase readily hydrolyzed casein, synthetic peptide butyloxycarbonyl-valine-leucine-lysine-~me~hylcouma~l-7-~ide (Boc-Val-LeuLys-MCA) and myosin heavy chain. The proteolytic activity was effectively inhibited by serine protease inhibitors such as soybean trypsin inhibitor and N,-p-tosyl+lysine-chloromethyl ketone, but was not affected by the other types of protease inhibitors (e.g. EDTA, EGTA, iodoacetic acid, mercuric chloride and iV-tosyl-L-phenylalanine-chloromethyl ketone). The optimal temperature was 40°C for hydrolysis of both casein and Boc-Val-Leu-Lys-MCA. The optimal pH values were 6.8 and 7.9 for caseinolytic and peptide hydrolyzing activity, respectively. The proteolytic activity was increased 1.3-fold by addition of @25M NaCl, but not by the addition of Ca2+. Myosin heavy chain was shown to be cleaved into smaller fragments by incubation with the proteinase on SDSPAGE. These results revealed that the enzyme was involved in degradation of myosin heavy chain from the squid mantle meat. Keywords: Myosin heavy chain degradation,

squid mantle muscle, trypsin-like

serine proteinase, thermally induced gel.

gels of spear squid Loligo bleekeri meat deteriorated and myosin heavy chains were degraded during precooking at 35°C (Nagashima et al., 1992). These phenomena were effectively suppressed by the addition of protease inhibitors such as ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) or soybean trypsin inhibitor, and indicated the implication of metalloand/or serine-proteinases in the textural deterioration of thermally induced gels from spear squid mantle meat. Tsuchiya and co-workers intensively investigated myosinases which specifically hydrolyzed myosin heavy chain, from Todarodes (Ommastrephes sloani) paczjicus mantle muscle (Okamoto et al., 1993). Little information, however, exists concerning serine proteinases in squid mantle meat. The objectives of this study were to purify serine proteinase responsible for myosin degradation from squid mantle muscle and to elucidate the cause of gel deterioration by pre-cooking.

INTRODUCTION Several studies have demonstrated that squid have various types of proteinases in their mantle muscle; acid cysteine proteinase, cathepsin D-like proteinase from Todarodes (Ommastrephes sloani) pacificus (Sakai & Matsumoto, 1981; Sakai-Suzuki et al., 1983, 1986), neutral metallo-proteinases from some of Teuthoidea (Ehara et al., 1992), and heat-stable alkaline protease from Loligo forbesi (Rodger et al., 1984). It is possible that these indigenous proteinases may be the cause of autolysis and subsequent textural destruction during storage, cooking or processing. We have previously reported that thermally induced

*Present address: Niigata Prefectural Fisheries Experimental Station, Igarashi, Niigata 950-21, Japan. ‘To whom correspondence should be addressed. 31

32

H. Ebina et al.

MATERIALS AND METHODS

4r

Materials Fresh spear squid Loligo bleekeri was purchased at the Tokyo Central Wholesale Fish Market, transported to the laboratory and immediately used for preparation. A frozen block of striped marlin Makaira mitsukurii was obtained from a local retail fish market. Protease inhibitors, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis-@minoethyl ether) N,N,N’,N’tetraacetic acid (EGTA), iodoacetic acid, mercuric chloride, phenylmethylstionyl fluoride (PMSF), soybean trypsin inhibitor, N,-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK) were obtained from Sigma Chemical Co. (St. Louis, MO). Synthetic fluorogenic peptide, butyloxycarbonyl-valine-leucine-lysine-4-methylcoumaryl-7amide (Boc-Val-Leu-Lys-MCA) was from Peptide Research Institute (Osaka, Japan). Molecular weight protein standards for SDS-PAGE were obtained from Sigma Chemical Co. All other chemicals were reagent grade.

Fractionnumber(5 ml/Fr.)

Fig. 1. Elution profile of the proteinase by Sephacryl S-400 column chromatography (second pass). Proteolytic activities were assayed as described in Materials and Methods. +, Caseinolytic activity; --O_-, Boc-Val-Leu-Lys-MCA-hydrolyzing activity, ---; absorbance at 280 nm. column (2.6 X 89 cm, Pharmacia LKB Biotechnology) equilibrated with 0.02 M phosphate buffer (pH 7-2), washed with the same buffer, and eluted by a linear gradient of O-O.8 M NaCl in the same buffer at a flow rate of 16 ml/h. All procedures for the preparation were conducted at 2-5°C in a cold room.

Purification of myosin heavy chain (MHC)degrading proteinase

Assay of proteolytic activity

After skinning, the mantle muscle of squid was minced with a prechilled chopper. The minced meat was homogenized with 3 volumes of 0.1 M KC1-0.02 M phosphate buffer (pH 7.2) containing 0.1% (v/v) Triton X-100 in a blender (Model AM-lo, Nihonseiki Co. Ltd, Tokyo, Japan) three times at 30-s-intervals. The homogenate was centrifuged at 10,000 g for 30 min and the resulting supernatant was collected as the crude extract for further purification. The crude extract was fractionated with solid ammonium sulfate at 30-&O% saturation. The precipitate was collected by centrifugation at 6,000g for 20 min, dissolved in a small volume of 0.1 M KC1-0.02 M phosphate buffer (pH 7.2) containing 0.1% (v/v) Triton X-100, and then dialyzed against the same buffer overnight. The dialyzate was further purified on a Sephacryl S400 column (5.8 X 54 cm, Pharmacia LKB Biotechnology, Bromma, Sweden) equilibrated with 0.2 M NaCl0.02 M phosphate buffer (pH 7.2) and eluted with the same buffer at a flow rate of 48 ml/h. Active fractions were collected and concentrated by ultrafiltration using a Diaflo PM-10 membrane (cut-off c lO,OOO-Da substances, Amicon, Danver, MA). The concentrate was rechromatographed on a Sephacryl S-400 column (2.6 X 95 cm) equilibrated with the same buffer at a flow rate of 18 ml/h. Active fractions (fraction numbers 67-103, Fig. 1) from the second Sephacryl S-400 column chromatography were pooled and concentrated by ultrafiltration in the same manner as described above. The concentrate was placed on a DEAE-Sepharose

Caseinolytic activity was determined as follows. The incubation mixture (1 ml of 2 mM CaCl,-O.2 M Tris-HCl buffer, pH 7.5, containing 10 mg of casein and 1 ml of enzyme solution (6 mg protein)) was incubated at 35°C for 60 min. The reaction was terminated by adding 2 ml of 5% (w/v) chilled trichloroacetic acid and centrifuged at 5500 g for 30 min. The amount of proteolytic products in the resulting supematant was measured by absorbance at 280 nm. One unit of caseinolytic activity was defined as the amount of enzyme that caused an increase of 0.1 absorbance unit at 280 nm after 60 min incubation at 35°C. The peptide-hydrolyzing activity was determined by using Boc-Val-Leu-Lys-MCA as a substrate. The incubation mixture (0.6 ml of 0.1 M Tris-HCl buffer, pH 7.5, containing 10 FM Boc-Val-Leu-Lys-MCA and 0.4 ml of enzyme solution (6 mg protein)) was incubated at 35°C for 60 min. The reaction was stopped by adding 1.5 ml of a 35 : 30 : 35 (v/v/v) cocktail of methyl alcohol : n-butyl alcohol: distilled water. The mixture was centrifuged at 5,500g at 5°C for 10 min. The amount of the liberated fluorogenic 7-amino-4-methylcoumarin (MCA) was measured at 435 nm with 367 nm excitation. One unit of the peptide-hydrolyzing activity was defined as the amount of enzyme that caused an increase of 0.01 fluorescence intensity after 60 min incubation at 35°C. The optimal temperature of the proteolytic activities was measured at pH 7.5 over a temperature range of 25 to 80°C. On the other hand, the optimal pH was measured at 40°C by using the following buffers: 2 mM

MHC-degrading

proteinase from spear squid muscle

CaCl,&2 M phosphate (pH 5.7 and 6. l), 2 mM CaCl,0.2 M Tris-HCl (pH 6.8, 7.6, 8.0 and 8.4), and 2 mM CaCl,-0.2 M carbonate (pH 8.5, 9.0 and 9.6) for caseinolytic activity; 0.1 M phosphate (pH 5.7 and 6. l), 0.1 M Tris-HCl (pH 6.8, 7.5, 7.9 and 8.2), and 0.1 M carbonate (pH 8.7, 9.2 and 9.7) for peptide-hydrolyzing activity. Preparation of myosin The dorsal muscle of partially thawed striped marlin was carefully excised. Myosin was prepared from dorsal muscle by the method of Mackie and Connell (1964) with some modifications. Myosin solutions were clarified by ultracentrifugation at 100,OOOgfor 30 min in the presence of 5 mM ATP and 5 mM MgCl,, and then myosin in the supernatant was collected by ammonium sulfate fractionation (given 40-55% saturation). Finally, the myosin was dialyzed against 0.6 M KClk@O2 M phosphate buffer (pH 7.5) at around 4°C overnight and subjected to the following analyses. Assay of MHC-degrading activity MHC-degrading activity was measured by SDS-PAGE analysis. Four millilitres of the proteinase solution in 0.02 M phosphate buffer (pH 7.2) were mixed with an equal volume of myosin solution (40 mg protein in 0.6 M KCl-0.02 M phosphate buffer, pH 7.5) from striped marlin muscle and then incubated at 35°C for up to 180 min. Each l-ml aliquot was taken out periodically and added to a solution containing 0.1 ml of 10% (w/v) SDS, 0.01 ml of 2-mercaptoethanol, 0.5 g of urea, and 0.2 M phosphate buffer, pH 7.0. The mixture was heated at 100°C for 2 min and dialyzed against 0.1% (w/v) SDS-O. 1% (v/v) 2-mercaptoethanol-00 1 M phosphate buffer (pH 7.0) at room temperature (around 18’C) for 12 h. The dialyzate was applied to SDSPAGE analysis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

33

RESULTS Purification of MHC-degrading proteinase The elution profile from the second Sephacryl S-400 column chromatography is illustrated in Figure 1. The activities of serine proteinase were determined using Boc-Val-Leu-Lys- MCA and casein as substrates. Two obvious peaks (fraction numbers 77 and 83) with peptidehydrolyzing activity were detected, while a major peak (fraction number 83) and a minor one (fraction number 56) with caseinolytic activity were observed. Active fraction (Fr. 1) was combined and further purified by DEAE Sepharose column chromatography. As shown in Figure 2, separation on DEAE sepharose column revealed three major peaks with activity toward Boc-Val-LeuLys-MCA; one peak in the unadsorbed fraction (Fr. II-a) and two peaks in the adsorbed fraction (Fr. II-b and -c), while a sharp peak with caseinolytic activity was observed in the unabsorbed fraction (Fr. II-a). Fr. II-a, which had both peptide-hydrolyzing and caseinolytic activities, showed a major protein band with a molecular weight of 42 kDa on SDS-PAGE (Fig. 2, inset). The proteinase thus obtained was used in the following analyses. Effects of temperature and pH Figure 3 shows that the proteinase was most active at 40°C toward both substrates, casein and Boc-Val-LeuLys-MCA at pH 7.5. The activities of the proteinase at 25°C were 30 and 69% of the maximum caseinolytic and Boc-Val-Leu-Lys-MCA hydrolyzing activities, respectively. On the other hand, at 60°C no peptide hydrolyzing activity was detected, while caseinolytic activity retained 36% of the maximum activity. 116 97.4

66 45

\d

29 kDa 4

Fr.IIa r

SDS-PAGE was conducted in the presence of 0.1% (w/v) SDS using 4 or 10% (w/v) polyacrylamide gels, according to the method of Weber and Osborn (1969), and gels were stained with Coomassie Brilliant Blue R250. The molecular weight of the proteinase was determined by using SDS-PAGE. P-Galactosidase (116 kDa), phosphorylase B (97.4 kDa), bovine albumin (66 kDa), egg albumin (45 kDa) and carbonic anhydrase (29 kDa) were used as standard proteins. Determination of protein concentration Protein concentration was determined by the biuret method (Gornall et al., 1949; Umemoto, 1966), using bovine serum albumin as a standard protein.

Fraction

number

(5 ml/Fr.)

Fig. 2. Elution profile of the proteinase by DEAE Sepharose

column chromatography. Proteolytic activities were assayed as described in Materials and Methods ----, Caseinolytic activity; --O-, Boc-Val-Leu-LysMCA hydrolyzing activity, ---; absorbance at 280 nm. Inset: SDS-PAGE of the proteinase (Fr. II-a) obtained from DEAE Sepharose column chromatography. A; molecular weight markers, B; the proteinase fraction (Fr.II-a).

H. Ebina et al.

34

Table 1. Effect of inhibitors on Boc-Val-LewLys-MCA hydrolyzing and caseh~olytic activities of the protehuwe Final concentration

Proteinase inhibitors

Relative activity (%) Boc-Val-Leu-Lys-MCA

Temperature (“C)

Fig. 3. Effect of temperature on proteolytic activities. --e--T Caseinolytic activity; 4, Boc-Val-Leu-Lys-MCA hydrolyzing activity.

None EDTA EGTA Iodoacetic acid Mercuric chloride PMSF TPCK* 1 TLCK*2 Soybean trypsin inhibitor

1.0 mM

1.0 rnM 1.0 rnM 1.0mM 1.0 mM

1.0 mM 1.0 rnM 1.0 mg/ml

100 84.5 87.1 85.5 79.6 87.3 90.5 3.1 1.3

Casein 100 81.5 83.1 76.3 93.2 100 108 46.3 0

* 1 N-tosyl+Phe-chloromethylketone. *2 N,-tosyl+Lys-chloromethylketone.

Addition of 0.25 M NaCl brought about a 1.3-fold increase in the proteolytic activity, but Ca2+ did not affect the activity (data not shown). MHCdegrading

PH

Fig. 4. Effect of pH on the proteolytic activities. a, seinolytic activity; --O, Boc-Val-Leu-Lys-MCA lyzing activity.

Cahydro-

The optimal pH values of the proteolytic activities were 6.8 and 7.9 for casein and Boc-Val-Leu-LysMCA, respectively (Fig. 4). Caseinolytic activity was considerably high at the neutral pH range, whereas Boc-Val-Leu-Lys-MCA hydrolyzing activity was reduced by 50% at pH 7 and had no activity below pH 6.

activity

Although attempts were made to prepare myosin from spear squid mantle muscle, the preparation was not successful without using inhibitors due primarily to instability of squid mantle muscle myosin during the preparation procedures (Konno, 1978; Tsuchiya et al., 1978). Therefore, myosin from striped marlin muscle was used instead of squid mantle muscle, since ‘modori’ (heat-induced gel degradation occurring especially at around 60°C) did not take place in muscle pastes from either species. As shown in Figure 5, MHC from striped marlin was apparently cleaved into smaller fragments by incubation with the proteinase. The MHC band was faintly detectable after 180 min incubation.

Effects of inhibitors The effects of various inhibitors on the proteolytic activities toward casein and Boc-Val-Leu-Lys-MCA are summarized in Table 1. Soybean trypsin inhibitor

and TLCK effectively inhibited both activities, while EDTA, EGTA (inhibitors of metallo-proteinases), iodoacetic acid and mercuric chloride (inhibitors of cysteine proteinases) barely possessed inhibitory effect. These results clearly suggest that the proteinase is a serine proteinase and might be a trypsin-like proteinase. We previously reported that EDTA, PMSF or soybean trypsin inhibitor inactivated indigenous proteinases in spear squid mantle meat (Nagashima et al., 1992, 1993). In this study, however, only soybean trypsin inhibitor was able to inhibit the activity of the purified proteinase.

A

B 0

AB 20 Incubation

AB 60

AB 180

time (min)

Fig. 5. Changes in SDS-PAGE patterns of striped marlin myosin by incubation with the proteinase. A, Myosin without the proteinase; B, myosin with the proteinase. MHC, myosin heavy chain.

MHC-degrading

proteinase from

spear squid muscle

35

Table 2. Comparison of biochemical properties of the trypsin-like proteinase and myosinases Trypsin-like proteinase Optimal pH

Myosinase I Myosinase II (Okamoto et al., 1993)

6,8*’

7.0*3

7.0*3

4o”c*3

4o”c*3

1,lO-Phenan throline, EDTA

l,lO-Phenan throline, EDTA, EGTA

Myosin heavy chain

Myosin heavy chain, tropomyosin, casein

16 kDa

20 kDa

1,9*=

Optimal temperature Inhibitors

Substrates

Molecular weight

400C*1.2 Soybean trypsin inhibitor*‘,= TLCK*‘,= Myosin heavy chain, casein, Boc-Val-Leu-Lys-MCA 42 kDa

*’ Casein was used as a substrate. *2Boc-Val-Leu-Lys-MCA was used as a substrate. *3Myosin heavy chain was used as a substrate.

The crude enzyme preparation degraded striped marlin MHC in a shorter time than the proteinase. In this case, MHC bands on SDS-PAGE completely disappeared after 180 min incubation (data not shown). It seemed that MHC-degradation products by the proteinase might have been further hydrolyzed by other proteinases contained in the crude enzyme preparation. As shown in Figure 2, at least two kinds of proteinases existed in the adsorbed fraction from DEAE Sepharose.

DISCUSSION

Our previous paper revealed that the metallo- and/or serine-proteinases degraded squid myosin and consequently deteriorated squid meat gel prepared by preheating at 35°C (Nagashima et al, 1992). The enzymic properties of the trypsin-like proteinase reported in this study strongly support the involvement of the proteinase in the deterioration of squid meat gel, due to the following: MHC-degrading activity, the optimal proteolytic activity at 40°C at the neutral pH, and increasing proteolytic activity by the addition of NaCl. Recent studies have indicated the existence of proteinases which hydrolyze myosin at neutral pH. Ehara et al. (1992) reported that myosin-degrading metalloproteinases were widely distributed in Teuthoidae mantle muscles and also found a metallo-proteinase in L. bleekeri. Novel metallo-proteinases, myosinases, were purified from Todarodes (Ommastrephes sloani) paczjicus, which degraded MHC with extremely high substrate specificity with optimal pH and temperature of 7.0 and 4O”C, respectively. As seen in Table 2, myosinases were, however, obviously distinct from the proteinase purified in this study in respect of inhibitory effect. Chymotrypsin and trypsin which are typical serine proteinases and specifically hydrolyze MHC at neutral pH, were distinguishable from the proteinase prepared

in the present study with regards to molecular weight, e.g. 25 kDa for chymotrypsin, 23.3 kDa for trypsin (Barman, 1985), and 42 kDa for the proteinase. In addition, MHC-degrading serine proteinases involved in ‘modori’ were found from several fish such as crucian carp (Kinoshita et al., 1990a), oval-filefish (Toyohara et al., 1990), threadfin-bream (Kinoshita et al., 1990b), and white croaker (Folco et al., 1989; Yanagihara et al., 1991). These MHC-degrading proteinases were most active at a temperature of 5060°C. MHC was also hydrolyzed by calpain which is a cysteine proteinase and requires Ca2+. Based on these results, the proteinase obtained from spear squid mantle muscle is a trypsinlike proteinase distinct from any MHC-degrading proteinases reported so far.

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Ebina et al.

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(Received 12 January 1994; accepted 20 May 1994)