characterization of proteolytic activity in octopus (octopus vulgaris) arm muscle

CHARACTERIZATION OF PROTEOLYTIC ACTIVITY IN OCTOPUS (Octopus vuZgds) ARM MUSCLE

JOSE LUIS HURTADO, JAVER BORDERhS, PLAR MONTERO

Instituto del Frio (C.S.I.C.) C i W Universitaria

28040 Madrid, SPAIN

AND

HAEJUNG AN'

Oregon State University-Seafood Laboratory 2001 Marine Dr., RM 253 Astoria. OR 97103-3427

Received for Publication May 19, 1998 Accepted for Publication December 10, 1998

ABSTRACT

A new proteolytic activity assay was devised to avoid the interference of paramyosin which causes gelling during the enzymatic assay. Extremely high autolytic activity was observed in octopus arm muscle, which was 40-500 fold nigher than those of various other fish species. The proteinase was inhibited strongly by leupeptin and iodoacetic acid and, to a lesser degree, by trans- epoxysuccinyl-L-leucy lamino (4-guanidono) butane (E-64), indicating the class as a thiolproteinase. The proteinase exhibited optimum activity at pH 2.5 and 40C, although it contained a suljhydryl group in the active site. Myosin heavy chain was the primary myojibrillar protein which was hydrolyzed during the autolysis of octopus arm followed by paramyosin. Actin showed no signs of hydrolysis during the incubation of up to 8 h. Due to its high afJinity for myosin, the enzyme activity should be controlled during processing octopus to ensure the functionality of myosin.

' Correspondence should be addressed to Dr. Haejung An, 2001 Marine Dr., RM 253, OSU- Seafood Laboratory, Astoria, OR 97103-3427, Phone: (503) 325-4531, Fax: (503) 325-2753, e- mail : haejung . an@orst.edu

Journal of Food Biochemistry 23 (1999) 469-483. All Rights Reserved. "Copyright 1999 by Food & Nutritwn Press, Inc.. Trumbull, Connecticut. 469

470 J.L. HURTADO, J. BORDERfAS, P. MONTERO and H. AN

INTRODUCTION

Octopus (Octopus vulgaris) is a popular cephalopod eaten as seafood in Mediterranean and Oriental countries. The cephalopod has a short life cycle, and is characterized by a high growth rate (Guerra 1992). Such rapid growth involves a high turnover rate of the body proteins, and thus intense proteolytic activities have been found in cephalopod muscle (Sakai and Matsumoto 1981). After death, cephalopods, i.e., octopus and squid, enter a state of high protein degradation by both endogenous and bacterial enzymes. Such rapid protein degradation results in release of high levels of nitrogen from the muscle, promoting bacterial growth leading to rapid decomposition. Thus, the shelf-life of octopus is extremely limited, typically 6-7 days after catch even at low storage temperature of 2.5C (Hurtado et al. 1997).

Although no information is available on proteolytic activity of octopus, some reports have been made on those of squid. Sakai and Matsumoto (1981) observed autolytic activity in the mantle muscle of Ommustrephes sloani pac@cus in the acidic pH range with the maximum at pH 3.1. Sakai et al. (1981) verified this acidic proteinase to have maximal proteolytic activity at pH 2.9 and the temperature of 35C and reported the presence of cathepsin D-like proteinases and a group of thiol-proteinases. The role of these thiol-proteinases was reported in a subsequent study by Sakai-Suzuki et al. (1983), in which the presence of a considerable amount of the acid thiol proteinase in the mantle muscle was detected with extracts prepared with DTT. About 39% of the DTT- activated acid proteinase activity was of an unknown nature and the squid mantle did not seem to contain thiol proteinases such as cathepsins B and L. Native myosin heavy chain was readily degraded by the acid thiol proteinase, while actin showed a little decrease in intensity of the band. Leblanc and Gill (1982) observed the majority of proteolytic activity to be in the acidic range of pH, which showed the maximal activity at pH 2.6 and 3.6 for ZZlex illecebrosus and Loligo pealei, respectively, and minor activity in the alkaline pH range. Based on the apparent molecular weight and optimal pH of the activity, they suggested that cathepsin D and E were the most active major proteinases from Loligo pealei and Illex illecebrosus, respectively. Hameed and Haard (1985) isolated and characterized cathepsin C from Illex illecebrosus. The enzyme was an octomer with a monomer molecular weight of 25 kDa and exhibited C1- and sulfiydryl dependence for catalysis. The pH profiles showed a biphasic nature for hydrolysis of substrate and inhibition by sulfiydryl enzyme inhibitors, i.e., iodoacetate, E-64, p-chloromercuribenzoate (p-CMB), and HgC1,. Other studies carried out by Konno and Fukazawa (1993) with Todaropsispacijicus, reported high autolytic activity which was effective in degrading myosin heavy chain (MHC) at the optimal condition of pH 7.0 and 40C. A rapid proteolysis of MHC was also observed by incubating Illex argentinus mantle at 35C

PROTEOLYTIC ACTIVITY IN OCTOPUS 47 1

(Kolodziejska er al. 1987). Although most of the activity was observed at temperature range 35-40C3, activity at higher temperature was also observed. Rodger et al. (1984) reported predominant proteolytic activity in Loligo forbesi at 60C in the alkaline range of pH with a maximum activity at pH 7.6. Ayensa (1997) reported two maximal autolytic activity peaks at 40 and 65C in squid mantle muscle (Todaropsis eblunae). The objective of this study was to characterize proteinase activity in octopus arm muscle, which consists of the edible main portion of the animal.

MATEFUALS AND METHODS

Samples

Octopus was harvested along the coast of Pontevedra in Spain and transported in ice to Instituto del Frio, (CSIC), Madrid, within 12 h postharvest. The animals were cleaned and the arms were collected, vacuum packed, and frozen at -4OC in an AGA-FRIGOSCANDIA freezer (model 0-6373). The octopus arm muscle was transferred frozen in dry ice to OSU-Seafood Lab, Astoria, OR, and kept at -8OC until used.

Preparation of Crude Extract

Frozen octopus arms weighing approximately 80-100 g were thawed, skinned, and manually chopped to approximate diameters of 3-4 mm. Due to extreme foaming, the muscle could not be homogenized using a blender or Polytron. The chopped octopus arm muscle was put in a mortar with acetone and dry ice. The volume of acetone used was minimal to cover the chopped muscle. The instantly frozen tissue was manually pulverized with a pestle. Once the muscle was comminuted into fine particles, acetone was evaporated under a stream of nitrogen gas. The homogeneous slurry was centrifuged (Sorvall RCSC with a rotor type SS-34) at 20,000 x g for 40 min at 4C, and the supernatant was used as a crude enzyme extract. The recovered enzyme extract was approximately 20-30% (v/w) of the original muscle used.

Autolytic Activity Assay

Three grams of finely chopped octopus arm muscle were spread in a thin layer in a 100-mL beaker and incubated in a water bath at 40C for 1 h. For temperature profile study, samples were incubated at 0-80C as specified in the text. The autolytic reaction was stopped by adding 15 mL of 10% (w/v) cold trichloroacetic acid (TCA). The mixture was incubated for 15 min at 4C and then centrifuged at 6,100 X g for 15 min to remove unhydrolyzed proteins. The TCA-soluble proteins were recovered from the supernatant and analyzed for

472 J.L. HURTADO, J. BORDERfAS, P. MONTERO and H. AN

oligopeptide content by the method of Lowry e? al. (1951). Samples were analyzed in duplicate. Autolytic activity was expressed as moles of tyrosine released per gram of muscle per hour (nmol Tyr/g/h).

Comparison of Autolytic Activities of Different Species

Autolytic activities in the muscle of octopus, squid (Turudores pacijicus), Chinook salmon (Onchorynchus mykiss), red rockfish (Sebasm sp.), mackerel (Scomber japonicus), and Pacific whiting (Merluccius productus) were tested at both 40 and 55C. The temperatures were chosen based on the highest autolytic activities observed €or octopus and fish muscles, respectively. Autolytic activity was analyzed as described in the previous section and expressed as nmol Tyrlgh.

Proteolytic Activity Assay

The crude enzyme extract contained a high level of protein, approximately 50 mg/mL, and was mainly composed of myofibrillar proteins. Due to the interference of paramyosin, which gelled during the activity assay and caused the precipitation ofprotein substrates, a modified activity assay method was used to analyze the activity in the crude extract. The crude extract (312.5 pL) was mixed with the same volume of McIlvaine’s buffer, pH 2.5 and incubated at 40C for 1 h. The incubation condition was chosen based on the optimum conditions observed for the autolytic activity. The reaction was stopped by adding 100 pL of 50% (w/v) TCA to the mixture. The mixture was then incubated at 4C for 15 min to allow precipitation of unhydrolyzed proteins, and they were removed by centrifugation at 5,700 x g for 10 min (Eppendorf Micro Centrifuge, Model 5415C, Brinkmann, New York). The TCA-soluble supernatant was analyzed for oligopeptide content by Lowry’s assay (Lowry e? al. 1951). The activity was expressed as nmol Tyr/gh.

pH Profile of Octopus Proteinases

Crude extract (312.5 pL) was added to an equal volume of the following buffers. McIlvaine’s buffer was used for the pH range of 1.5-8.0, and Tris-HC1 (0.2 M) for pH 8.5-10.0. The mixture was incubated at 40C for 1 h, and the reaction was terminated by adding cold (4C) TCA to the mixture. The hydrolyzed oligopeptide content was analyzed as described in the section “Proteolytic activity assay”.

Chemical Inhibition

All four types of inhibitors, i.e., serine, cysteine, aspartic acid and metallo- proteinase, were tested to determine the class of the proteinase in crude extract.

PROTEOLYTIC ACTIVITY IN OCTOPUS 473

The inhibitors tested included ethylenediamine-tetraacetic acid (EDTA), phenanthroline, E-64, p-CMB, iodoacetic acid, leupeptine, phenylmethylsulfonyl fluoride (PMSF), trypsin inhibitor, and pepstatin. Inhibitor stock solutions (100 pL) were added to crude extract (312.5 pL) to give the final concentrations listed in the text and the mixture was preincubated for 15 min at room temperature. MacIlvaine’s buffer, pH 2.5 was added to the mixture, and the residual activity was analyzed as described in the section “Proteolytic activity assay”.

Substrate Specificity

Various protein substrates, i.e., hemoglobin (Hb) denatured in 0.06 N HC1, 1% (w/v) casein, 1% (w/v) bovine serum albumin (BSA), and 1% (w/v) azocasein, were compared for the hydrolytic efficiency of the octopus arm muscle proteinase(s) . Stock solutions of the protein substrates were prepared at 1 % (w/v), and 2 mg substrate was added to 625 pL of McIlvaine’s buffer, pH 2.5. The reaction mixture was adjusted to the total volume of 850 pL with water and preincubated at 40C for 1 min. Crude enzyme extract, 400 pL, was added to the mixture and incubated for 1 h at 40C. The reaction was stopped by adding 200 pL of cold 50% (w/v) TCA solution to the mixture. After the addition of TCA, the mixture was incubated at 4C for 15 min to precipitate unhydrolyzed proteins and centrifuged at 5,700 x g for 10 min (Eppendorf Micro Centrifuge, Model 5415C, Brinkmann, New York) to remove the precipitates. The hydrolyzed oligopeptides in the supernatant released from the protein substrates were estimated by Lowry’s method (Lowry ef al. 1951). Activity was expressed as change in absorbance (AA750) as compared to a blank. For azocasein, hydrolyzed products were estimated by the method of An ef al. (1994a) by monitoring increase in absorbance at 450 nm compared with that of a blank (AA.450).

Sample Preparation for Gel Electrophoresis

Octopus arm muscle was finely chopped manually and one gram of the chopped muscle was incubated at 40C in a water bath for various periods, i.e., 0 min (Control group), 30 min, 1, 2, 4 and 8 h, to induce various degrees of autolysis. To the control and autolyzed samples, 9 mL of solubilization buffer (20 mM Tris-HC1, pH 8.0 containing 2% SDS, 2% 8-mercaptoethanol, and 8 M urea) was added and the mixtures were stirred continuously with a magnetic stirring bar overnight at room temperature (approximately 22C) to solubilize total proteins. The solubilized homogenates were centrifuged at 10,OOO x g (Sorvall RCSC, DuPont Co., Newtown, CT) for 20 min at room temperature to remove the undissolved tissue debris. The supernatant was analyzed €or protein concentration by Lowry’s assay (Lowry ef al. 1951), and 60 pg proteins

474 J.L. HURTADO, J. BORDERfAS, P. MONTERO and H. AN

were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE

Gel electrophoresis was carried out according to Laemmli (1970) using 10% polyacrylamide gels. Proteins were separated at the constant current of 15 mA for stacking gels and 30 mA for separating gels. The separated proteins were fixed and stained with 0.125% Coomassie brilliant blue R-250 in 25% ethanol and 10% acetic acid and destained in 25% ethanol and 10% acetic acid. High molecular weight standard markers (Sigma Chemical Co., St. Louis, MO) were used to assess molecular weights of proteins. They contained rabbit myosin (M, 205,000), 0-galactosidase (M, 116,000), phosphorylase b (M, 97,000), fructose- 6-phosphate kinase (M, 84,000), bovine albumin (M, 66,000), glutamic dehydrogenase (M, 55,000), ovalbumin (M, 45,000), and glyceraldehyde-3- phosphate dehydrogenase (M, 36,000).

To quantitate protein degradation, SDS-PAGE gels were scanned using an HP Scan Jet I1 scanner (Hewlett-Packard Co., Minneapolis, MN). Intensity of myosin, paramyosin and actin bands were estimated as area under the peak using NIH Image Software 1.54 (NIH, Washington, DC).

RESULTS AND DISCUSSION

Comparison of Autolytic Activities in Muscle of Various Marine Species

Two cephalopods and four fish species were tested to compare autolytic activities in their muscle. Both cephalopods, i.e., octopus and squid, showed higher autolytic activities than fish species at both temperatures tested, 40 and 55C (Table 1). Our results showed that autolytic activities of cephalopods were higher, in general, at 40C, while fish had higher autolytic activities at 55C. At 40C, the autolytic activity of octopus was 2.8 fold that of squid; 26.7 fold that of Pacific whiting which showed the highest activity among fish; and 481.1 fold that of salmon, showing the lowest activity. At 55C, the activity of octopus was decreased to 19.6% that of 40C. At that temperature, the activity level was 67.4% that of squid. Compared to fish, the activity of octopus was about 1.7- 38.4 fold higher at 55C. Ayensa (1997) compared autolytic activities in squid (Todaropsis eblanae) and blue whiting (Micromesistius poutassou) and reported that the activity observed for squid was higher than that of blue whiting for all the incubation temperatures tested, 0-90C. Other investigators also have reported high endogenous proteolytic activity in other species of cephalopods (Sakai and Matsumoto 1981; Leblanc and Gill 1982; Konno and Fukazawa 1993).

PROTEOLYTIC ACTIVITY IN OCTOPUS 475

TABLE 1 . AUTOLYTIC ACTIVITY OF MARINE SPECIES AT 40 AND 55C

Activitv’

Species 40C 55c

Squid octopus Mackerel Salmon Rockfish

Pacific whiting

376.56 1058.44

25.93 2.22

14.46 39.64

307.95 207.55 119.15 25.83 5.39

71.16

’ Autolytic activity was expressed as nmol Tyr/g/h.

Hydrolytic Efficiency for Various Protein Substrates

No significant differences in hydrolytic activity of octopus proteinase were observed with casein, Hb and BSA used as substrates as shown by the narrow range of absorbance observed, 0.202-0.263 (Table 2). The crude extract used as a source of enzyme contained a large amount of proteins, as shown by the protein content, approximately 50 mg/mL. Among the components, myosin was shown to be the most preferred substrate and was the first protein hydrolyzed by the proteinase(s) as discussed in the later section “Degradation Pattern of Octopus Muscle”, thus showing the minimal effect of added exogenous substrates.

TABLE 2. PROTEOLYTIC ACTIVITY OF CRUDE EXTRACT WITH DIFFERENT SUBSTRATES

AT 40C AND DH 2.5

Substrates AAbs’

Azocasein Casein Hb BSA

0.093 0.263 0.202 0.233

~~

‘Absorbance was read at 428 nm for azocasein and 750 nm for casein, Hb and BSA.

476 J.L. HURTADO, J. BORDERfAS, P. MONTERO and H. AN

Amcasein, often used as a good substrate for analyzing proteinase activity in fish muscle (An et al. 1994a), was not suitable for octopus proteinase(s) and resulted in extremely low readings, (A4=, 0.093, Table 2). Again, the presence of high levels of myofibrillar proteins in the crude extract interfered with the assay by the competitive inhibition of the proteinase, resulting in substantial reduction in hydrolysis of azocasein. An et al. (1994a) reported that high levels of proteins included in the extract may compete with azocasein for the active site of proteinases, thus underestimating the enzyme activity.

Temperature Profile of Autolytic Activity

Autolytic activity in octopus arm muscle was studied over the range of 0- 80C. The activity gradually increased until it reached a peak at 40C (Fig. 1) followed by a rapid decrease to 55C. At temperatures above 60C, the activity increased gradually to 8OC, but no obvious peak was found in this temperature range. It is unlikely the increase in absorbance was due to a proteinase. The two most heat-stable proteinase groups present in fish muscle are known as cathepsin L and alkaline proteinase (An et al. 1996). However, even these proteinases were rapidly heat-inactivated at temperatures above 70C (Makinodan ef al. 1987; Boye and Lanier 1988; Stoknes et al. 1993; Seymour et al. 1994). Therefore, it is postulated that the apparent increase in released tyrosine was due to nonspecific hydrolysis of muscle proteins rather than proteolytic release of peptides from the muscle. Since the maximum autolytic activity was observed at 40C for 0. vulgaris, this temperature was chosen for activity analysis in further analyses. Sakai et al. (1981) reported the presence of acid proteinases in crude extract of squid (Omstrephes sloanipac@cus) with maximum activity at 35-40C followed by a substantial decrease above 45C. Konno and Fukazawa (1993) found maximal autolysis at 40C in squid (Todurodes pacificus) mantle muscle, followed by a decrease at temperatures above 40C with no activity found above 50C. However, Ayensa (1997) reported two maximal activity peaks at 40 and 65C in squid mantle muscle (Toduropsis eblanae), as analyzed at pH 7.0. Rodger et al. (1984) have reported proteolytic activity at 60C in squid muscle (Loligoforbesi) using casein as a substrate, which showed an optimum activity at pH 7.6.

pH Profile of Proteolytic Activity

The pH-activity profile demonstrated a high level of proteolytic activity in the acidic pH range from 2.0 to 4.5, with the optimum found at pH 2.5 (Fig. 2). No activity was detected in the pH range above 6.0 on the assay condition of 40C. The presence of acid proteinase has been previously reported in squid mantle muscle. Sakai et al. (1981) observed the maximal activity of squid mantle muscle at pH 2.9 and 35C. Leblanc and Gill (1982) reported the

PROTEOLYTIC ACTIVITY IN OCTOPUS 477

FIG. 1. AUTOLYTIC ACTIVITY OF OCTOPUS ARM MUSCLE MEASURED AT VARIOUS TEMPERATURES

The activity is expressed in nmol Tyr/g/h. Each point represents the mean of duplicate measurements.

n 80 bl e 70 M 2

E" 6o

z 8 40

.a 2o

b o

50

a4 30

a4

v1 g 10

A

1 2 3 4 5 6 7 8 9 1 0

FIG. 2. EFFECT OF pH ON PROTEOLYTIC ACTIVITY OF OCTOPUS ARM MUSCLE The activity was assayed at 40C for 1 h. The activity is expressed nmol Tyr/g/h. Each point

represents the mean of duplicate measurements.

478 J.L. HURTADO, J. B O R D E R h , P. MONTERO and H. AN

maximal activities at pH 2.6 and 3.6 in the mantle muscle of Zllex illecebrosus and Loligopealei, respectively, although other activities were also evident in the alkaline pH range. On the contrary Konno and Fukazawa (1993) reported the maximal autolysis of T. paczjicus mantle muscle at pH 7.0.

Effect of Proteinase Inhibitors on Activity

The proteolytic activity was inhibited most strongly (80%) by leupeptin. About 50% inhibition was obtained with iodoacetic acid and E-64. Leupeptin, iodoacetic acid, and E-64 inhibit thiol proteinases; thus, inhibition of the octopus proteinase by these compounds indicated the presence of a group of thiol- proteinases (Table 3). Sakai et al. (1981) have demonstrated the presence of thiol proteinases in an acid pH range of 2.9 in squid mantle muscle at 35C. The authors reported that, although the activity was inhibited by both iodoacetic acid and leupeptin, the highest inhibition, 76.3 %, was achieved with pepstatin. Sakai- Suzuki et al. (1983) observed a considerable amount of acid-thiol proteinases in squid mantle muscle when extracted with D'M', whose optimum activities were found at pH 2.9 and 35C. The authors obtained the higher rate of inhibition, 61 % , of the extract by combining both thiol and acid proteinase inhibitors, i.e., iodoacetic acid and pepstatin. Ayensa (1997) has reported 80% inhibition of squid (Todaropsis eblanae) mantle muscle proteinase in acid range @H 4.5 and 5.5) with E-64. Nagashima et al. (1992) used EDTA, PMSF and soybean trypsin inhibitor in the inhibition assay and proposed that metallo- and serine proteinases were most likely involved in deterioration of squid (Loligo bleekei) meat gels heated at 35C. Although leupeptin has been commonly used to inhibit thiol cathepsins such as cathepsin B (Aoyagi et al. 1969) and cathepsin L from rat liver and rabbit skeletal muscle (Kirschke et al. 1977; Okitani et al. 1980), Sakai-Suzuki et al. (1983) could not detect the presence of such cathepsins in squid mantle muscle. The authors reported no activity hydrolyzing N-benzoyl- DL-arginine-p-nitroanilide (BAPA) and azocasein with the reaction time up to 17 h. In comparison, many investigators demonstrated the degradation of myofibrillar proteins in fish was due to the activity of thiol cathepsins (Yamashita and Konagaya 1991; Morrissey et al. 1993; An et al. 1994a).

Degradation Pattern of Octopus Muscle

The SDS-PAGE electrophoregram of octopus arm muscle showed the presence of myosin heavy chain (MHC), paramyosin (PM), actin (A), and two myosin light chains (MLC) in the decreasing order of molecular weights, as previously reported by Kariya et al. (1986) (Fig. 3). After 30 min incubation at 40C, the bands corresponding to MHC disappeared with the appearance of three new bands at the lower molecular region. Konno and Fukazawa (1993) reported that MHC of squid gradually disappeared when incubated at 25C and the

PROTEOLYTIC ACTIVITY IN OCTOPUS 479

TABLE 3. EFFECT OF PROTEASE INHIBITORS ON PROTEOLYTIC ACTIVITY OF

OCTOPUS ARM MUSCLE

Inhibitor Concentration

Control EDTA Phenanthroline

Iodacetic acid Leupeptin p-Chloromercuribenzoate PMSF Trypsin inhibitor Pepstatin

E-64

none 10 mM

1 mg/mL 10 mM 1 m M 1mM

0.01 mM 1 mglmL 2 mglmL 1 mg/mL

% Residual Activity

100 78.29 65.18 53.40 54.18 22.80 78.46 72.01 91.31 17.28

FIG. 3. SDS-PAGE PATTERN OF OCTOPUS ARM MUSCLE INCUBATED AT 40C FOR VARIOUS TIME PERIODS

Lanes: (1) Molecular weight standards, (2) Control, (3) 30 min, (4) 1 h, (5) 2 h, (6) 4 h, and (7) 8 h. Protein bands are marked for myosin heavy chain, MHC; paramyosin, PM; and actin,

A. The bands of protein standards are marked on left.

480 J.L. HURTADO, J . BORDERfAS, P. MONTERO and H. AN

complete disappearance was noted in 4 h. Initially, 72% of the PM band was hydrolyzed rapidly within 30 min incubation at 40C, but 12% of the residual intensity was still detected at 8 h incubation as estimated by densitometric scanning (Table 4). Actin was not affected by 8 h incubation, showing only 13 % hydrolysis compared to the original intensity of the band.

TABLE 4. THE PERCENT RESIDUAL CONTENTS' OF MYOFIBRILLAR PROTEINS

DURING AUTOLYSIS

proteins Control 30min l h 2 h 4 h 8 h

Myosin 100 0 0 0 0 0 Paramyosin 100 27.9 14.0 13.3 16.7 11.6 Actin 100 > 100 91.7 > 100 > 100 87.2

' Octopus muscle samples were autolyzed at 40C for the designated period of time and subjected to electrophoresis. Percent residual contents of myofibrillar proteins were estimated by scanning SDS-PAGE gel for area under the peak.

These results imply that the proteinases play a major role in the degradation of octopus arm muscle proteins. MHC seem to be the main target of this proteinase(s), followed by PM. Sakai e? al. (1981) first showed that several myofibrillar proteins of the squid mantle muscle, including MHC and PM, were most rapidly degraded at pH 3.1, where thiol-proteinases might contribute to the degradation of the proteins. Sakai-Suzuki ef al. (1983) reported severe degradation of squid mantle myofibrils when incubated at pH 2.5. At this pH, degradation of MHC was more intense with thiol-proteinases than cathepsin D- like proteinases, although cathepsin D-like proteinase could degrade MHC. The authors also reported the possibility of thiol-proteinases degrading actin, but in this study we did not observe degradation of the actin band with 8 h incubation at 40C. It has been reported that actin is highly resistant to degradation by muscle proteinases (An et al. 1994b; Wasson e? al. 1992).

SUMMARY

A high level of proteolytic activity was detected in cephalopods, octopus in particular, compared to various species of fish. Octopus arm proteinase showed similar properties to that of squid muscle. Proteolytic activity of octopus muscle

PROTEOLYTIC ACTIVITY IN OCTOPUS 48 1

exhibited a pH optimum in an acid range at pH 2.5 and temperature optimum at 40C. The activity was inhibited strongly by leupeptin, and to a lesser degree by iodoacetic acid and E-64, indicating the presence of thiol-proteinase(s). MHC was the primary target during autolysis of octopus arm muscle, followed by paramyosin. Actin showed no signs of hydrolysis by the proteinase during the incubation period studied.

ACKNOWLEDGMENT

We wish to thank the Education and Science Ministry, Spain for providing a Formation Scholarship of Personal Investigation to JosC Luis Hurtado, Instituto del Frio (C.S.I.C.), Ciudad Universitaria, Madrid, Spain.

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