Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas

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1 Blackwell Science, LtdOxford, UK FISFisheries Science Blackwell Science Asia Pty Ltd 691February 2003 fistest.doc Denaturation and autolysis of jumbo squid K Konno et al /j fistest.doc.x Original Article204209BEES SGML FISHERIES SCIENCE 2003; 69: Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas Kunihiko KONNO, 1, * Cho YOUNG-JE, 2 Takeya YOSHIOKA, 1,3 Park SHINHO 1 AND Nobuo SEKI 1 1 Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido , 3 Hokkaido Industrial Technology Center, Hakodate, Hokkaido and 2 Department of Food Science and Technology, Pukyong National University, Pusan , Korea ABSTRACT: Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca 2+ -, Mg 2+ - and K + (EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca 2+ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25 C. Washing the homogenate partially reduced the autolysis activity. KEY WORDS: myofibrils. ATPase activity, autolysis, calcium ion, inactivation, jumbo squid, thermal INTRODUCTION Myosin from Japanese common squid Todarodes pacificus has unique properties in its thermal denaturation profile. When heated as myofibrils, Ca 2+ -ATPase inactivation proceeded very slowly in the presence of Ca 2+ compared with that with EDTA. The stabilization of myosin by Ca 2+ was approximately 100-fold. 1 Stabilization by Ca 2+ was also detected with myosin subfragment-1(s-1), but the extent was remarkably less. 2 In other words, myosin stabilization by Ca 2+ was fully achieved when bound to F-actin. Another characteristic property of squid myofibrils in thermal denaturation was that the inactivation rate was remarkably increased by increasing the KCl concentration for heating. 3 Inactivation was accelerated by fold when KCl concentration for heating was raised from 0.1 M to 0.8 M, at which the maximal rate was obtained. This profile indicated that squid myosin in myofibrils under a physiological ionic strength was strongly stabilized by F-actin. 4 It is well known that squid mantle muscle contains proteolytic enzymes that degrade myosin molecules into shorter fragments. It is also *Corresponding author: Tel: Fax: konno@fish.hokudai.ac.jp Received 18 April Accepted 5 September believed that the degradation of myosin by the enzymes leads to deterioration of the thermal gel. We have previously reported that mantle muscle of common squid contains at least two types of proteolytic enzyme: one cleaves myosin molecule into heavy meromyosin (HMM) and light meromyosin (LMM) and the other cleaves myosin into subfragment-1 (S-1) and rod. 5 Metalloprotease, the enzyme responsible for the former activity, has been purified from the mantle muscle of common squid and well characterized. 6 Common squid is still the major species captured in Japan. However, other species of squid, especially frozen imported squid, have been used for manufacturing various types of products. We have reported a species-specific thermal stability of myofibrils from eight species of squid and cuttlefish and showed that all of the myofibrils were well stabilized upon addition of calcium ion. 3 Distribution of proteolytic activities among squid and cuttlefish has been studied carefully and it was concluded that the enzyme is distributed widely in the mantle muscle of many species of squid, but not in cuttlefish. 7 A new species of squid has been brought into Japan as a substitute of common squid. Jumbo squid, which belongs to the same family of Ommastrephidae as common squid, is one of these. There is no information on the properties of its muscle proteins. For its utilization, it seems to be essential to understand the biochem-

2 Denaturation and autolysis of jumbo squid FISHERIES SCIENCE 205 ical and denaturation profiles of myofibrils and autolysis profiles of the mantle muscle of jumbo squid. In the present paper, we investigated the biochemical properties of myofibrils of jumbo squid by measuring three types of ATPase activities, thermal inactivation profile of myofibrillar Ca 2+ -ATPase and the autolysis profile by monitoring the cleavage of myosin upon the incubation of muscle homogenate of jumbo squid. MATERIALS AND METHODS Jumbo squid Disidicus gigas used was captured off the coast of Peru and stored frozen at - 40 C. Samples stored for about 6 months were used in the study. Samples had a length of approximately cm, a weight of 5 6 kg and a thickness of cm for mantle muscle. Myofibrils were prepared from by applying the same procedures used for myofibril preparation from common squid. 1 Mantle muscle was repeatedly homogenized with 0.1 M KCl and 20 mm Tris-HCl (ph 7.5) and the homogenate was washed by repeating the suspension and centrifugation. Ca 2+ -, Mg 2+ - and K + (EDTA)-ATPase activities of the myofibrils were measured as has been reported previously. 8 The thermal denaturation rate for the myofibrils was studied by measuring the Ca 2+ -ATPase inactivation upon heating. Incubation was conducted with either 1 mm CaCl 2 or EDTA. 1 KCl concentration for heating was varied optionally. The thermal inactivation rate of Ca 2+ -ATPase was the indicator for the thermal denaturation of myofibrils. Ca 2+ -ATPase was assayed as reported previously. 1 To study the effect of NH 4 Cl on the thermal inactivation of jumbo squid myofibrillar Ca 2+ -ATPase, various concentrations of NH 4 Cl were further added to the myofibril suspended in 0.1 M KCl and 20 mm Tris- HCl (ph 7.5). The autolysis profile of jumbo squid mantle muscle was studied by applying the same procedures used for common squid. 5 Muscle homogenate was used as a model system. Mantle muscle (5 g) was homogenized with 45 ml of 0.1 M NaCl and 20 mm Tris-HCl (ph 7.5) for 30 s at r.p.m with a blender (Nihon Seiki, Tokyo, Japan). Blending was repeated four times. Homogenate was filtered through a layer of cotton gauze. The filtrate (ª 8 10 mg/ml) that could be handled quantitatively was incubated under various conditions for studying the autolysis. NaCl concentration for the incubation medium and incubation temperatures was changed optionally. Autolysis rate was estimated by measuring the heavy chain content detected on sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). 9 In the estimation, the reaction was assumed to obey the first-order reaction. RESULTS AND DISCUSSION ATPase activities of jumbo squid myofibrils Three types of ATPase activities of jumbo squid myofibrils were measured by changing the KCl concentration. The results are shown in Fig. 1. Ca 2+ -ATPase activity showed maximal activity at approximately M KCl with specific activity of approximately 1.2 mmol Pi/min per mg. Such a high specific activity was almost the same as that of common squid myofibrils. Suppression of myosin Ca 2+ -ATPase activity by F-actin in low-salt medium was also detected with jumbo squid. 10 At low KCl concentrations, a high Ca-sensitivity was detected in Mg 2+ -ATPase; high activity with Ca 2+ and low activity without Ca 2+. However, the sensitivity gradually decreased with increasing KCl concentration as a result of a gradual decrease in the activity with Ca 2+ and a gradual increase in the activity without Ca 2+. Only a very low Ca-sensitivity was detected above 0.5 M KCl. It should be noted Fig. 1 Three types of ATPase activities of jumbo squid myofibrils. ( ) Ca 2+ -ATPase, ( ) Mg 2+ -ATPase (+ Ca), ( ) Mg 2+ -ATPase ( Ca) and ( ) K + (EDTA)-ATPase activities were measured as a function of KCl concentrations at 25 C. Assay medium for Ca 2+ -, Mg 2+ - and K + (EDTA)- ATPase contained 5 mm CaCl 2, 2 mm MgCl 2 and 1 mm EDTA, respectively. Medium for Mg 2+ -ATPase (+ Ca), Mg 2+ -ATPase ( Ca) assay also contained 0.1 mm CaCl 2 and 0.5 mm ethylene glycol bis (b-aminoethylether)- N,N,N,N -tetraacetic acid (EGTA), respectively. The medium always contained 20 mm Tris-maleate (ph 7.0) and 1 mm ATP.

3 206 FISHERIES SCIENCE K Konno et al. that Mg 2+ -ATPase activities above 0.3 M KCl were as high as approximately 0.1 mmol Pi/min per mg, indicating that Mg 2+ -ATPase of myosin alone without activation by F-actin is quite high because actin activation above 0.3 M KCl was negligible with common squid myosin. 11 These profiles of Mg 2+ -ATPase activities were the same as those of common squid myofibrils. 8 K + (EDTA)-ATPase activity at 1 M KCl was as low as 0.02 mmol Pi/min per mg, which was similar to that of common squid myofibrils. These results demonstrated that jumbo squid myofibrils were practically indistinguishable from common squid myofibrils. Thermal denaturation profile of myofibrils of jumbo squid Thermal denaturation profile of common squid myofibrils was characterized by stabilization upon addition of Ca 2+. We studied whether thermal denaturation of myofibrils of jumbo squid was also stabilized by Ca 2+. As we had no information on the thermal stability of the myofibrils of jumbo squid itself, we studied the thermal stability of jumbo squid myofibrils. To assess it, the myofibrils were heated at various temperatures for 30 min in the presence of either 1 mm CaCl 2 or EDTA. It was demonstrated that no inactivation was detected below 30 C even with EDTA, and an almost complete inactivation at 45 C even with CaCl 2 was detected. We found that a suitable temperature for studying the stabilizing effect of Ca 2+ on jumbo squid myofibrils was 35 C. This temperature was the same as that for studying the effect of Ca 2+ with common squid myofibrils. Thus, the thermal stability of jumbo squid myofibrils was concluded to be very similar to that of common squid myofibrils. Inactivation profiles of Ca 2+ -ATPase for jumbo squid myofibrils at 35 C with 1 mm Ca 2+ and EDTA are presented in Fig. 2. Inactivation was very slow when the medium contained Ca 2+ with an inactivation rate of /s, while addition of EDTA significantly accelerated the inactivation with a rate of /s. The increase in the inactivation rate upon addition of EDTA was more than 60-fold. It was concluded that stabilization by Ca 2+ was almost the same with jumbo squid myofibrils as for common squid myofibrils. Next, we studied how KCl concentration affects the thermal inactivation rate of jumbo squid myofibrils. Results are presented in Fig. 3. With increasing KCl concentration for heating, the inactivation rate in the presence of both Ca 2+ and EDTA increased and reached a plateau at around 0.7 M KCl where stabilization by F-actin would be lost. 12 Increase in the KCl concentration from 0.1 to 0.8 Fig. 2 Effect of Ca and EDTA on the thermal inactivation profiles of jumbo squid myofibrillar Ca 2+ -ATPase. Myofibrils from jumbo squid were suspended in 0.1 M KCl, 20 mm Tris-HCl (ph 7.5) and heated at 35 C with either ( ) 1 mm CaCl 2 or ( ) EDTA. Thermal inactivation profile of Ca 2+ -ATPase was followed. Fig. 3 Effect of KCl concentration on the Ca 2+ -ATPase inactivation rate of jumbo squid myofibrils. Myofibrils were suspended in media containing various concentrations of KCl. The media also contained either ( ) 1 mm CaCl 2 or ( ) EDTA. The suspension was heated at 35 C and the inactivation rates were estimated. increased the inactivation rate from to /s in Ca 2+ medium with 480-fold acceleration, and from to /s in EDTA medium with an acceleration of only 14-fold.

Denaturation and autolysis of jumbo squid FISHERIES SCIENCE 207 Apparent stabilization by Ca 2+ at 0.7 M KCl medium where myosin lost the stabilization by F-actin was about twofold.

2 It is concluded that significant stabilization by Ca 2+ would be achieved only when myosin was fully stabilized by F-actin under physiological ion strength.

4 Denaturation and autolysis of jumbo squid FISHERIES SCIENCE 207 Apparent stabilization by Ca 2+ at 0.7 M KCl medium where myosin lost the stabilization by F-actin was about twofold. This small extent of stabilization was very similar to that for myosin subfragment-1 of common squid. 2 It is concluded that significant stabilization by Ca 2+ would be achieved only when myosin was fully stabilized by F-actin under physiological ion strength. It is well established with fish myofibrils that thermal denaturation of myofibrils was strongly affected by the ph for incubation, and myofibrils are most stable at neutral ph. 13 Myofibrils of jumbo squid were heated at 35 C in medium containing 0.1 M KCl and either 50 mm Tris-maleate (ph 5.5 7) or Tris-HCl (ph 7.5 8). The minimum inactivation rate was at ph 7.0, the same ph reported with fish myofibrils. 13 It is well known that mantle muscle of jumbo squid contains NH 4 Cl as high as 50 mm, although its physiological function is unclear. We wondered whether NH 4 Cl affects the thermal inactivation rate of jumbo squid myofibrils. Myofibril in 0.1 M KCl, 20 mm Tris-HCl (ph 7.5) and 1 mm CaCl 2 was heated with various concentrations of NH 4 Cl. The thermal inactivation rate of myofibrils with an additional concentration of KCl was also estimated as control. As shown in Fig. 4, addition of NH 4 Cl increased the inactivation rate slightly, but the increase was the same as that caused by KCl addition. It was concluded that NH 4 Cl at the concentration range of M caused no significant effect on the inactivation rate of jumbo squid myofibrils. Autolysis profiles of jumbo squid mantle muscle Strong protease activities in the mantle muscle of common squid caused serious damage to gel formation of squid meat. We wondered whether jumbo squid contains such protease activities. We investigated the autolysis profile of jumbo squid mantle muscle by using muscle homogenate instead of muscle itself for a quantitative analysis of autolysis. Muscle homogenate was prepared by blending mantle muscle with 9 volumes of 0.1 M NaCl, 20 mm Tris-HCl (ph 7.5). The intact protein components of jumbo squid mantle muscle could be seen for the sample without incubation (0 min in Fig. 5). As the homogenate contained watersoluble components in addition to myofibrillar Fig. 4 Effect of NH 4 Cl and KCl concentration on the Ca 2+ -ATPase inactivation rate of jumbo squid myofibrils. Various amounts of either ( ) NH 4 Cl or ( ) KCl as indicated in the horizontal axis were added to myofibrils (0.1 M KCl, 20 mm Tris-HCl (ph 7.5), 1 mm CaCl 2 ), and incubated at 35 C. Fig. 5 Autolysis profile of jumbo squid mantle muscle. Muscle homogenate of jumbo squid in 0.5 M NaCl, 20 mm Tris-HCl (ph 7.5) was incubated for up to 4 h at 25 C. 4ED is the incubated sample for 4 h with 1 mm EDTA. The degradation profile of myofibrillar proteins was analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HC, PM, Actin are myosin heavy chain, paramyosin and actin, respectively. HMM-HC and LMM are autolysis products of heavy meromyosin heavy chain and light meromyosin, respectively.

5 208 FISHERIES SCIENCE K Konno et al. proteins, the electrophoretic pattern was not simple. Myosin heavy chain and actin bands are two major components noticeable in the pattern. Although the data are not presented, the SDS- PAGE pattern for the myofibril of jumbo squid was indistinguishable from that for the myofibrils of common squid. A component supposed to be paramyosin was detected in the pattern. Incubation of homogenate at 25 C in 0.5 M NaCl resulted in the degradation of myosin heavy chain into two fragments (Fig. 5). The short fragment had a similar mobility to paramyosin. This cleavage pattern was indistinguishable from that of common squid. 5 In common squid, the fragments indicated as HMM- HC (heavy meromyosin heavy chain) and LMM in Fig. 5 are proved to be HMM and LMM, respectively. 5 We examined whether two fragments produced from jumbo squid myosin are the corresponding ones. The long fragment was recovered in the water-soluble fraction when centrifuged with ATP-Mg, indicating that the fragment is HMM like. The short fragment was recovered in the saltsoluble fraction irrespective of the presence of ATP, indicating that the fragment is LMM like (data not shown). It was thus concluded that the autolysis profile of jumbo squid mantle muscle was essentially identical to that of common squid. When the medium contained 2 mm EDTA, the autolysis was significantly suppressed (4ED in Fig. 5). These results indicated that the proteolytic enzyme involved in the autolysis of jumbo squid is metalloprotease, the same as that detected in the mantle muscle of common squid. As the autolysis rate is strongly dependent on the protein concentration, 5 direct comparison of the autolysis rate between jumbo squid and common squid is difficult. However, when the rate at a similar protein concentration of 5 6 mg/ml was compared, jumbo squid homogenate gave a similar rate to that of common squid homogenate. Jumbo squid used in the present study was stored frozen. Nevertheless, the autolysis rate was similar to that of fresh common squid. It was suggested that the proteolytic enzyme was kept active during the frozen storage for at least 6 months. Effect of NaCl concentration on the autolysis was examined. It is established that autolysis is greatly affected by the state of myosin in homogenate. 5,14 Homogenates were incubated at 25 C for 4 h in the presence of various concentrations of NaCl, and the myosin heavy chain content remaining was estimated on SDS-PAGE. The autolysis rate was calculated by assuming that the reaction obeys the first-order reaction. Figure 6 shows the results. Autolysis at approximately 0.1 M NaCl was not obvious, while the rate increased with increasing NaCl concentration, showing a maximal rate at Fig. 6 Effect of NaCl concentration on the autolysis rate of jumbo squid mantle muscle. The same homogenates as in Fig. 5 were used. The homogenates were incubated at 25 C at various concentrations of NaCl. The autolysis rate was estimated by measuring the disappearing rate of myosin heavy chain in the sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) pattern. Fig. 7 Temperature-dependent autolysis rate of jumbo squid mantle muscle. Muscle homogenate was incubated at various temperatures from 10 to 40 C, and the disappearing rate of myosin heavy chain was measured. The other conditions were the same as in Fig. 5. approximately 0.3 M NaCl. Above this concentration, the rate gradually decreased. The rate at M NaCl was roughly half that at 0.3 M NaCl. This NaCl concentration-dependent autolysis rate was

6 Denaturation and autolysis of jumbo squid FISHERIES SCIENCE 209 practically indistinguishable from that of common squid. A low autolysis rate in low salt medium would be due to protection of the cleavage site, HMM/LMM junction, by self-association to form filaments. 14 We then examined how the autolysis rate is accelerated when the incubation temperature is changed. Homogenate dissolved in 0.5 M NaCl was incubated at temperatures from 10 to 40 C. The autolysis rates estimated are presented in Fig. 7. The rate increased with raising the temperature from 10 to 25 C; however, the rate drastically decreased above 30 C indicating that the enzyme responsible for the autolysis was readily inactivated above this temperature. Almost no autolysis was detected at 40 C. These results suggested that the proteolytic enzyme contained in jumbo squid muscle is quite unstable. We tested whether the proteolytic enzyme could be removed from the homogenate. We used a simple method of washing with 0.1 M NaCl, 20 mm Tris-HCl (ph 7.5). Homogenate was diluted with 9 volumes of the buffer and centrifuged. The residue was repeatedly suspended and centrifuged for removing water-soluble fraction from the homogenate. Once washed, the homogenate showed the reduced rate by about one-half, but further repeated washings only slightly reduced the rate. The sample that was washed four times showed a rate roughly one-third that of the unwashed sample. These results indicated that complete removal of protease was not achieved by the above washing procedures. Although the data are not presented, the autolysis profile as studied by SDS-PAGE of fourfold the washed sample was the same as that for the unwashed sample, indicating that the remaining enzyme is the same as the major protease involved in the autolysis of squid mantle muscle, the metalloprotease. In conclusion, jumbo squid mantle muscle was indistinguishable from that of common squid in the biochemical properties and thermal inactivation profile of myofibrillar Ca 2+ -ATPase and autolysis profile. In other words, jumbo squid could be used as a raw material similar to common squid without special precautions. Hokkaido University and Pukyong National University. REFERENCES 1. Konno K. Thermal denaturation of squid myofibrils. Effects of calcium ion and EDTA. Nippon Suisan Gakkaishi 1991; 57: Konno K. Complex formation between regulatory and essential light chain on squid mantle myosin subfragment- 1 revealed by thermal denaturation method. J. Biochem. 1991; 109: Konno K, Yuasa M. Comparative aspect of thermal stability of squid myofibrils. Comp. Biochem. Physiol. 1993; 105A: Wakameda A, Nozawa S, Arai K. Effect of neutral salts on thermal denaturation of myofibrillar Ca-ATPase of fish. Nippon Suisan Gakkaishi 1983; 49: Konno K, Fukazawa C. Autolysis of squid mantle muscle protein as affected by storage conditions and inhibitors. J.Food Sci. 1993; 58: Okamoto Y, Otsuka-Fuchino H, Horiuchi S, Tamiya T, Matsumoto JJ, Tsuchiya T. Purification and characterization of two metalloproteases from squid mantle muscle, myosinase I and myosinase II. Biochim. Biophys. Acta 1993; 1161: Ehara T, Tamiya T, Tsuchiya T. Investigation of myosin heavy chain degrading proteinase in Decapoda muscle. Nippon Suisan Gakkaishi 1992; 58: Yoshitomi B, Konno K. Enzymatic properties of myosin ATPase from squid Todarodes pacificus mantle muscle. Nippon Suisan Gakkaishi 1982; 48: Porzio MA, Pearson AM. Improved resolution of myofibrillar proteins with sodium dodecylsulfate-gel electrophoresis. Biochim. Biophys. Acta 1977; 490: Konno K, Yoshitomi B. Interaction of squid myosin with F- actin in low-salt medium. Nippon Suisan Gakkaishi 1981; 47: Konno K, Arai K, Yoshida M, Watanabe S. Calcium regulation in squid mantle and scallop adductor muscles. J. Biochem. 1981; 89: Wakameda A, Arai K. The denaturation mechanism of carp myosin B in the presence of high concentration of salts. Nippon Suisan Gakkaishi 1984; 50: Hashimoto A, Arai K. The effect of ph and temperature on the stability of myofibrillar Ca-ATPase from some fish species. Nippon Suisan Gakkaishi 1978; 44: Konno K, Nakajima A, Koseki H, Sakai T. Effects of sorbitol on the autolysis profiles of squid mantle muscle. Fish. Sci. 2002; 68: ACKNOWLEDGMENTS This study was conducted as part of the Core University Program on Fisheries Sciences between

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