Blackwell Science, LtdOxford, UKFISFisheries Science0919-92682005 Blackwell Publishing Asia Pty LtdApril 2005712405413Original ArticleThermal denaturation pattern of fish myosinM Takahashi et al.
FISHERIES SCIENCE
2005; 71: 405–413
Species-specific thermal denaturation pattern of fish myosin when heated as myofibrils as studied by myosin subfragment-1 and rod denaturation rates Masayuki TAKAHASHI, Takeshi YAMAMOTO, Sanae KATOa and Kunihiko KONNO* Laboratory of Marine Food Sciences, Graduate School of Fisheries Science, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
ABSTRACT: Thermal denaturation of myofibrils from various species of fish was investigated by measuring ATPase inactivation, myosin aggregation, myosin subfragment-1 (S-1) and rod denaturation rates as studied by chymotryptic digestion. Decrease in monomeric myosin (myosin aggregation) was always faster than the ATPase inactivation for all myofibrils tested. The relative denaturation rate of rod to that of S-1 differed from species to species. Preceded denaturation of rod was observed with some species, and the opposite was true with other species. The denaturation pattern was explained by the different magnitude of S-1 stabilization by F-actin in myofibrils at low salt medium. Myofibrils which receive a great stabilization by F-actin as studied by ATPase inactivation showed the preceded rod denaturation pattern, and vice versa. S-1 portion, not F-actin, determined the different stabilization of S-1 by F-actin in myofibrils. KEY WORDS: F-actin, myofibril, myosin, species specificity, thermal denaturation.
INTRODUCTION Myosin serves as the most important role in the thermal gelation process in fish meat. One of the characteristic properties of fish muscle protein, especially myosin, is its unstable nature.1 Thus, many efforts were devoted to elucidate the myosin denaturation process during the storage and processing of fish meat, and to develop effective additives to prevent myosin denaturation for long-term storage of fish meat without loss of quality. The successful additives to prevent myosin denaturation are sugar and sugar alcohol, which are major cryoprotectants used for frozen surimi.2 Myosin molecule has a unique structure consisting of two 200 kilodalton (kDa) heavy chains (HC) and two sets of two types of light chain (LC) components with sizes of about 20–27 kDa.3 Amino terminal 100 kDa of a single HC together with two types of LC forms water soluble globular, subfragment-1 (S-
*Corresponding author: Tel: 81-138-40-5567. Fax: 81-138-40-5567. Email:
[email protected] a Present address: Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. Received 14 May 2004. Accepted 14 October 2004.
1), and the rest of two HC subunits forms salt soluble coiled-coil rod structure. The former, head portion of myosin molecule contains ATPase and F-actin binding sites, and the latter, tail region, assembles to form filaments under physiological conditions of low salt. Among the biochemical functions of myosin, ATPase activity is widely used as an indicator for detecting myosin denaturation. This allows us to analyze myosin denaturation quantitatively by assuming the inactivation process as the first order reaction mechanism.4–6 By analyzing the thermal inactivation of Ca2+-ATPase of myofibrils from various species of fish, it was concluded that thermal stability of fish myosin is deeply dependent on the inhabiting water temperature.7,8 The Ca2+-ATPase was also used as a useful indicator for evaluation of the quality of frozen surimi.9 Ca2+-ATPase inactivation provides sufficient information on the denaturation of the S-1 portion because ATPase active site resides there, but there is no information on the denaturation of rod portion. Studies on the thermal denaturation of rod was made with isolated fish myosin rod, and a similar conclusion to that with S-1 was proposed that myosin rod isolated from cold water species is more unstable than that from warm water species.10,11 In our previous studies with carp myosin and myofibrils, we found two unusual phenom-
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ena.12–14 The first one was that myosin forms aggregate at the neck region before loosing ATPase activity, indicating an important role of the neck region in the myosin aggregation process. The other was obtained with myofibrils that rod denaturation precedes S-1 denaturation. The latter was an unexpected conclusion because the isolated rod was much more stable than S-1. A question may be raised whether the event that rod denatures faster than S-1 found with carp myofibrils is generally observed with other species of fish. In the present paper, we investigated the myosin denaturation mode when heated as myofibrils from several species of fish by measuring S-1 and rod denaturation rates. MATERIALS AND METHODS Myofibrils were prepared from the dorsal muscle of the following species of fish as Katoh et al. described.9 Live carp Cyprinus carpio, rainbow trout Oncorhynchus mykiss, and tilapia Tilapia mossambica, as well as fresh yellowtail Seriola quinqueradita, pink salmon Oncorhynchus gorbuscha, brown sole Pleuronectes herzensteini, witch flounder Glyptocephalus stelleri, atka mackerel Pleurogrammus azonus, and walleye pollack Theragra chalcogramma were all purchased at local markets. Minced muscle was homogenized in 0.1 M KCl, 20 mM Tris-HCl (pH 7.5), and washed repeatedly with the same buffer. Washed myofibril suspension was finally filtered through two layers of gauze to remove connective tissues, and the filtrate was used as myofibril suspension. Thermal denaturation of myofibrils was studied in the above buffer. Heating temperature was varied from species to species so as to make the thermal denaturation mode analysis easier. The indicators employed for detecting myosin denaturation upon heating of myofibrils were as follows: (i) Ca2+ATPase inactivation; (ii) myosin aggregation as measured by the decrease in the monomeric myosin content which was measured by using ammonium sulfate fractionation at 40% saturation in the presence of 2 mM Mg-ATP; and (iii) amounts of chymotryptic fragments produced and monomeric ones when digested at either S-1/Rod or heavy meromyosin/light meromyosin (HMM/ LMM) junctions. The amount of myosin and chymotryptic fragments were estimated by measuring the staining intensity of the corresponding bands on sodium dodecylsulfate–polyacrylamide gel eletrohoresis (SDS-PAGE) using SHIMADZU dual wavelength flying spot scanning densitometer CS9300 PC (Shimadzu, Kyoto, Japan). SDS-PAGE was performed according to Laemmli.15
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Inactivation rates of myofibrillar Ca2+-ATPase at varied KCl concentrations were studied as Wakameda et al. reported.16 The walleye pollack actocarp S-1 hybrid sample was prepared by the S-1 exchange technique. First, myosin in walleye pollack myofibrils was cleaved into S-1 and rod by chymotrypsin at 10∞C by using 1/500 (w/w) of chymotrypsin over myofibrils for 30 min. The digest was heated at 30∞C for 30 min in the presence of 1 mM Mg-pyrophosphate to achieve a complete inactivation of S-1 in the digest. Mg-pyrophosphate contained in the medium removed the protective effect of F-actin on S-1 resulting in a quick denaturation of S-1. Carp S-1 was separately isolated from myofibrils as we have established. The amount of carp S-1 added was 1/5 (w/w) that of the digest. The amount was smaller than the S-1 content in walleye pollack digest. This was because all of the carp S-1 added can bind to F-actin leaving no free S-1. The mixture was dialyzed against 0.1 M KCl, 20 mM Tris-HCl (pH 7.5) to allow carp S-1 to bind to walleye pollack F-actin remaining in the digest upon removal of Mg-pyrophosphate. Denatured walleye pollack S-1 was not removed from the system, but it seemed unaffected in the results. RESULTS AND DISCUSSION Thermal denaturation mode of myosin in walleye pollack myofibrils The first fish species we examined was walleye pollack, the most important species for surimi production. Walleye pollack myofibrils in 0.1 M KCl, 20 mM Tris-HCl (pH 7.5) were heated at 30∞C, and their Ca2+-ATPase inactivation and myosin aggregation profiles were compared (Fig. 1a). A half inactivation of Ca2+-ATPase occurred in 22 min, while monomeric myosin content decreased faster with a half life of 12 min indicating that walleye pollack myosin formed aggregates before losing its ATPase activity when heated as myofibrils. The event was qualitatively the same as observed with carp myofibrils as previously reported.13 With carp myofibrils, a quick drop of monomeric myosin content was explained by a quick denaturation of rod portion of myosin upon heating.13 We wondered whether the same mechanism is applicable to the event with walleye pollack myofibrils. To answer the question, the denaturation rates of S-1 and rod upon heating of myofibrils were studied by using the chymotryptic digestion technique. Heated myofibrils were digested with chymotrypsin at the S-1/rod junction of myosin molecule in the absence of divalent cation (0.05 M KCl, 20 mM Tris-maleate [pH 7.0], 1 mM EDTA). The
Thermal denaturation pattern of fish myosin
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Fig. 1 Thermal denaturation of walleye pollack myofibrils. Walleye pollack myofibrils suspended in 0.1 M KCl, 20 mM Tris-HCl (pH 7.5) were heated at 30∞C. (a) Ca2+ATPase inactivation (open circles) and monomeric myosin content (open triangles) were estimated. (b) Amount of monomeric subfragment-1 (closed circles), rod produced (open triangles) and monomeric rod produced (closed triangles) were estimated after chymotryptic digestion of the heated myofibrils.
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digests were subjected to ammonium sulfate fractionation at 40% saturation with 1 mM Mg-ATP to recover monomeric S-1 and rod in the supernatant. The amounts of S-1 and rod produced and monomeric ones were both estimated by SDSPAGE. Results are presented in Fig. 1b. Upon heating of myofibrils, both S-1 and rod production decreased with duration. The decrease in the amount of rod produced from the heated myofibrils was slower than that of S-1 produced. We found that the amount of S-1 decreased at the same rate as the ATPase inactivation (compare Fig. 1a,b). This was a confirmation that S-1 was produced only from active myosin remaining in myofibrils, and S-1 of inactivated myosin was degraded into short fragments. Heating for 60 min reduced the S-1 production to 25% of that of the unheated sample, while rod production was kept as high as 85%. Ammonium sulfate fractionation revealed that all of S-1 produced was monomeric. However, some of the rod produced was present as aggregates. Yield of monomeric rod derived from the 60 min heated myofibrils (55%) was still higher than monomeric S-1(25%). A slow decrease of the amount of rod produced and monomeric rod than S-1 was characteristic for walleye pollack myofibrils. As we have reported, rod denaturation preceded the S-1 denaturation with carp myofibrils.13 These results showed that the denaturation mode with walleye pollack myofibrils was opposite to that of carp myofibrils, and that the denaturation mode as expressed by the S-1 and rod denaturation rates studied with carp myofibrils is not always applicable to other species of fish, and that the thermal denaturation mode was suggested to differ from species to species.
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Comparison of the thermal denaturation mode of myofibrils among fish species We further studied the thermal denaturation mode with other species of fish myofibrils by measuring the S-1 and rod denaturation rates. The species examined are rainbow trout (at 30∞C), brown sole (at 30∞C), pink salmon (at 35∞C), yellowtail (at 40∞C), tilapia (at 40∞C), atka mackerel (at 30∞C), and witch flounder (at 35∞C). Figure 2 shows the three typical patterns of S-1 and rod denaturation profiles obtained. We found that decreases in the amount of S-1 and rod produced could be well analyzed by assuming the first order reaction mechanism giving a straight line when logarithmic S-1 and rod content was plotted against the heating time. The denaturation rates of S-1 and rod were calculated from the slopes of the lines obtained. The first pattern was obtained with witch flounder (Fig. 2a). Duration of witch flounder myofibrils at 35∞C reduced the amount of S-1 produced. However, the heating did not reduce the amount of rod produced; the heated myofibrils for 60 min produced almost a similar amount of rod, while the production of S-1 was reduced to only about 13%. Although the digest contained a small amount of aggregated rod as revealed by ammonium sulfate fractionation, the decrease in the amount of monomeric rod was still very slow (the rate relative to S-1 decrease was about 0.1). Thus, it was demonstrated that rod portion of witch flounder myosin was hard to be denatured when heated as myofibrils. The denaturation mode of yellowtail was clearly different from that of witch flounder (Fig. 2b). The decreasing rate of rod production was slower than S-1 with the relative rate of 0.4,
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which was qualitatively the same as observed with witch flounder in Fig. 2a, while the decreasing rate of monomeric rod was practically identical to that of S-1. If one takes the amount of monomeric rod as an index of native rod referring to aggregated rod as denatured rod, denaturation mode of yellowtail myofibrils was characterized by the same denaturation of rod with S-1. We also found another denaturation pattern with rainbow trout myofibrils (Fig. 2c). The amount of rod produced and monomeric rod decreased at the same rate which was 2.6 times greater than S-1 decrease (Fig. 2c). The pattern was characterized by the absence of aggregated rod although the amount of rod decreased very quickly. The profile obtained with rainbow trout myofibrils was practically the same as observed with carp myofibrils. Above results demonstrated that the pattern differed from species to species. The denaturation modes for all of the species studied are summarized in Fig. 3, in which rod denaturation rates relative to S-1 are presented. Rod denaturation rates were estimated by following the decreasing rate of the amount of rod production and monomer rod as conducted in Fig. 1b. We also separately measured the myosin aggregation rate for the species of fish myofibrils as in Fig. 1a. Even though the three typical denaturation modes were presented in Fig. 2, the mode could not be classified into the above three groups, and dispersed greatly. The relative rod denaturation rates varied from 2.8 with carp to 0.1 with witch flounder. Studying the results shown in Fig. 3, we found that all rod produced was monomeric for the species whose rod denaturation rate was greater than S-1 (carp, rainbow trout, brown sole, and pink salmon), and that rod contained aggregate with the
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Fig. 2 S-1 and rod denaturation profiles for three typical species of fish. Myofibrils from witch flounder (a, 30∞C), yellowtail (b, 40∞C) and rainbow trout (c, 35∞C) were heated and decreasing of monomeric subfragment-1 (closed circles), rod produced (open triangles) and rod monomer (closed triangles) were estimated as in Fig. 1b.
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Fig. 3 Relative rod denaturation rates for fish species. Denaturation rates of rod were estimated as in Fig. 1b by measuring the amount of rod produced (shaded column) and monomeric rod (open column). The rates were expressed as ratios to the subfragment-1 denaturation rate. Monomeric myosin decreasing rates (closed circles) were also estimated as in Fig. 1a. Fish species and heating temperatures employed are carp (a, 40∞C), rainbow trout (b, 30∞C), brown sole (c, 35∞C), pink salmon (d, 35∞C), yellowtail (e, 40∞C), tilapia (f, 40∞C), atka mackerel (f, 30∞C), walleye pollack (h, 30∞C), and witch flounder (i, 35∞C)
species whose rod denaturation rate was smaller than S-1 (yellowtail, tilapia, atka mackerel, walleye pollack, and witch flounder). We tried to find the rule in the rod denaturation rate for species. The first factor we considered was the species-specific thermal stability. The heating temperatures presented in the legend of Fig. 3 would provide rough information on the thermal stability of myofibrils;
Thermal denaturation pattern of fish myosin
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species heated at higher temperatures are stable species, and vice versa. The species were arranged in order of the rod denaturation rate relative to S-1. No rules could be seen in the appearance of the heating temperatures. Thus, the thermal stability of myofibrils was not involved in the specific rod denaturation rate. Marine and freshwater species did not explain the distribution either. As brown sole and witch flounder showed an opposite denaturation mode, it was not true that the similar species showed a similar profile. Myosin aggregation rates relative to the ATPase inactivation rate, corresponding to the S-1 denaturation rate are presented in the same figure (Fig. 5). The rates differed from species to species. A clear rule was found that myosin aggregation rates were the same with rod denaturation rates with species whose rod denaturation was greater than S-1 (carp, rainbow trout, brown sole, and pink salmon). It was concluded that, as reported with carp myofibrils in the previous paper,13 the rod denaturation process as proved by chymotryptic digestion was the rate-limiting process of myosin aggregation for the species. Probably, myosin formed aggregates at rod portion, and rod portion for the aggregated myosin was susceptible to chymotryptic attack. The explanation was not applicable to the rest of the species in which the rod denaturation rate was equal or smaller than S-1. The myosin aggregation rate for the species was always about 1.5 times greater than the S-1 denaturation rate irrespective of the rod denaturation rate. As the myosin aggregation was always faster than S-1 denaturation, S-1 denaturation could not explain the event either. Neither S-1 nor rod denaturation explained the fast aggregation of myosin for the latter group including walleye pollack. We further studied the mechanism for the latter case by digesting myofibrils at the HMM/LMM junction instead of the S1/rod junction. The digestion was performed in the medium containing 0.5 M KCl, 20 mM Tris-HCl (pH 7.5) and 1 mM CaCl2. Change in the digestion patterns of walleye pollack myofibrils upon heating are presented in Fig. 4. As has been reported, unheated walleye pollack myosin was fairly selectively cleaved into HMM and LMM with sizes of 160 kDa and 70 kDa, respectively.17,18 Digestion pattern for walleye pollack myosin was different from that of carp myosin, of which rod contained several cleavage sites within producing several species of HMM and LMM.11 The digest was also subjected to the aggregation test using ammonium sulfate fractionation. The amount of fragments estimated from the pattern presented in Fig. 4 are shown in Fig. 5. The amount of HMM produced decreased gradually with duration at 30∞C. We
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Fig. 4 Change in the sodium dodecylsulfate–polyacrylamide gel electrohoresis (SDS-PAGE) pattern of the chymotryptic digest of walleye pollack myofibrils. Walleye pollack myofibrils as heated in Fig. 1 were digested in the presence of 1 mM CaCl2 in 0.5 M KCl so as to cleave myosin into heavy meromyosin (HMM) and light meromyosin (LMM). SDS-PAGE patterns for (a) the digest and (b) monomeric components in the digest were presented. Act, actin.
found a part of HMM produced was present as aggregates giving a little faster decreasing rate of monomeric HMM than HMM produced. This was completely different from the case of S-1, which was all recovered in the supernatant at 40% saturation as monomer. In the same figure (Fig. 5), ATPase inactivation and decrease in monomeric myosin content (myosin aggregation) profiles were also presented. Decreasing rate of HMM production was the same as ATPase inactivation, which was the same event as observed with S-1 production, namely active myosin generated HMM as well as S-1. The decrease of monomeric HMM was a little faster than ATPase inactivation, and was the identical to myosin monomer decrease. These results suggested that HMM as well as myosin formed aggregates before losing its ATPase activity. The characteristic structure common in myosin and HMM but not in S-1 and rod was the neck structure constructed by connecting S-1 and rod. Thus, the aggregation of active walleye pollack myosin in myofibrils was suggested to occur at the neck region. We have reported the myosin aggregation at the region before losing the activity when carp myosin in 0.5 M KCl was heated.14 We proposed in the paper that thermal movement of myo-
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The factor that determined the species-specific thermal denaturation mode
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Fig. 5 Decrease in heavy meromyosin (HMM) and light meromyosin (LMM) upon heating of walleye pollack myofibrils. Walleye pollack myofibrils were heated and digested as in Fig. 4. The amount of HMM produced (open triangles), monomeric HMM (closed triangles), LMM produced (open squares), and monomeric LMM (closed squares) were estimated from the patterns in Fig. 4. Ca2+-ATPase inactivation (closed circles) and monomeric myosin (open circles) decrease were taken from Fig. 1a.
sin tail functioned in the aggregate formation by active myosin at the neck region. In the present study, heating of myofibrils was conducted in a medium of 0.1 M KCl containing no ATP, where myosin rod portion associated to form filaments and S-1 bound to F-actin strongly. It is hard to imagine a similar free movement of myosin tail to associate at the neck with the myofibrils under the conditions. Weakened myosin/myosin and actin/ myosin interaction might make such reaction to occur with the species whose rod denaturation was slow. Increased flexibility of the myosin tail at high temperature would be confirmed by the fact that cleavage sites within rod increased at higher temperatures.17,19 Practically no decrease in LMM production upon heating of myofibrils could be seen; LMM portion was not degraded into much shorter fragments. Moreover, no aggregated LMM was detected in the digest (Figs 4,5). The portion seemed to be kept native throughout the heating process.
We concluded that the thermal denaturation mode of S-1 and rod was species-specific. However, we were uncertain what factor determined the pattern. It is well established that myosin or S-1 is remarkably stabilized upon forming a complex with F-actin which resulted in the reduction of inactivation of ATPase. As the heating of myofibrils was conducted at 0.1 M KCl, the thermal inactivation rates estimated in the present paper are those of myosin in a stabilized form by F-actin. Wakameda et al. carefully studied the effect of salt concentration on the thermal inactivation rate of myofibrils, and concluded that increase in the salt (NaCl, KCl) concentration accelerates the thermal inactivation rate as a result of loss of protection by F-actin.16 They also showed that the magnitude of acceleration in the ATPase inactivation rate upon increasing the salt concentration differs from species to species. We investigated how differently KCl concentration affects the thermal inactivation rates of ATPase for carp and walleye pollack myofibrils. In good agreement with their results, thermal inactivation rate of myofibrils increased with increasing the KCl concentration, and reached the maximal rate at around 1.5 M KCl for both species (Fig. 6). Wakameda et al. reported that the maximal inactivation rate of myofibrils at around 1.5–2 M KCl corresponded to the rate of myosin alone without stabilization by F-actin.20 Although the data are not presented, we confirmed it by comparing the inactivation of carp myofibrils at 2 M with that of isolated myosin. These results indicated inversely that myosin gradually gained the protection by F-actin by lowering the salt concentration. This would be reasonable because increased salt concentration reduces the affinity of myosin to Factin, which was demonstrated by the reduced activation of actin-activated S-1 Mg2+-ATPase activity.21 We compared the extent of stabilization achieved by F-actin at 0.1 M KCl by taking the change in the rate obtained by shifting KCl concentration from 0.1 M to 1.5 M between carp and walleye pollack. We noticed that the extent of stabilization for carp (about 30 times) was clearly greater than that for walleye pollack (about 10 times). It was demonstrated that myosin in walleye pollack myofibrils was weakly stabilized than that in carp myofibrils. By using the results, we led the conclusion differently from Wakameda et al.16 that stabilization of myosin by F-actin is speciesspecific. Magnitudes of stabilization by F-actin were estimated for six species of fish including carp and walleye pollack by performing the same set of
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Fig. 6 KCl concentration dependent thermal inactivation rate of carp and walleye pollack myofibrils. Thermal inactivation rates for carp (a, 35∞C) and walleye pollack (b, 25∞C) myofibrils were measured by changing the KCl concentrations.
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experiments as above. The obtained data were used for constructing Fig. 7 where the relative rod denaturation rate was plotted against the extent of stabilization by F-actin for respective species. A fairly good positive relationship was obtained
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between these two. As the ATPase resides at S-1 portion, the stabilization by F-actin as measured by the ATPase inactivation rate indicates how strongly S-1 portion of myosin is stabilized upon Factin binding. This relationship in Fig. 7 indicated that the greater the stabilization of S-1 portion is, the faster the rod denaturation becomes. The results seemed conflicting, but the relationship would be explained as follows. We assume two myofibrils having the same inactivation rate at 2 M KCl, namely the same denaturation rate of S-1 portion, and the same denaturation rate of rod portion, but different magnitude of stabilization by F-actin. Such myofibrils show different ATPase inactivation rates at 0.1 M KCl for two samples. To induce the same degree of ATPase inactivation (S-1 denaturation) at 0.1 M KCl, the species with greater stabilization require much severe thermal treatment than those with small stabilization. As we assumed the same denaturation rate of rod portion for two species, denaturation extent for the former one was much greater than that for the latter one. Different magnitude of stabilization by F-actin between carp and walleye pollack Regarding the different extent of stabilization of myosin by F-actin, we next studied which determined the stabilization extent, myosin or F-actin. A direct answer could be obtained by studying the thermal inactivation of the reconstituted hybrid actomyosin preparations using myosin and actin from carp and walleye pollack. Preparative methods of walleye pollack myosin22 or S-123 has been established, however, they require an addition of a
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high concentration of sorbitol or sodium glutamate to prevent their denaturation during the preparation and storage because of its low stability. We did not employ the method because we did not like to consider the side-effects of these additives. Instead, we employed the S-1 exchange method; S1 in walleye pollack myofibrils was exchanged by carp S-1, leaving rod portion of walleye pollack myosin and F-actin in the myofibrils. Myofibrils of carp and walleye pollack were digested by chymotrypsin, and their thermal inactivation rates at varied KCl concentrations are in Fig. 8. The cleavage practically unaffected the extent of stabilization by F-actin indicating that stabilization by F-actin did not involve the rod portion (Fig. 8). We noticed that KCl concentration required for a complete loss of protection decreased for carp myofibrils by the digestion. KCl concentration of 1 M KCl was high enough to remove the protection completely for the digest. This might be due to the reduced affinity of S-1 to F-actin compared with that of myosin to F-actin. Walleye pollack digest required the same concentration for the loss of protection as intact myofibrils. We have no explanation for the difference. Probably, intrinsic low affinity of S-1 to Factin with walleye pollack might be involved in the case. To exchange S-1 in walleye pollack digest by carp S-1, the following tricky treatments were conducted. Walleye pollack digest was added by 1 mM Mg-pyrophosphate (PPi) to remove the protection by F-actin24 and heated at 30∞C for 60 min to achieve a complete denaturation of S-1. After cooling the digest, carp S-1 was added to it. To allow carp S-1 to rebind to walleye pollack F-actin in the digest, Mg-PPi in the mixture was removed upon
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Fig. 8 KCl concentration dependent thermal stability of hybrid acto-subfragment-1 (S-1) complex. KCl concentration dependent inactivation rates were estimated for myofibrils of carp (closed circles in a) or walleye pollack (closed circles in b), digest of myofibril of carp (open circles in a) or walleye pollack (open circles in b). Hybrid walleye pollack acto-carp S-1 (open triangles in a) was constructed by using the techniques described in the text.
dialysis. The dialyzate still contained inactivated walleye pollack S-1, but its bad effect on the binding of carp S-1 was not detected probably due to a complete loss of binding ability for the denatured walleye pollack S-1. Then, we prepared hybrid walleye pollack acto-carp S-1 leaving the other components unchanged. We studied the KCl concentration dependent thermal inactivation rate for the hybrid preparation. It is established that the thermal inactivation rate of reconstituted actomyosin is determined by the myosin species not Factin.25 In agreement with the report, the hybrid preparation showed the same thermal inactivation rate as carp myofibrils requiring the heating temperature of 35∞C not 25∞C used for intact walleye pollack myofibrils (Fig. 8). The hybrid preparation showed the identical KCl concentration dependency not only in the magnitude of stabilization but also in the concentration for inducing a full removal of protection by F-actin; 30 times stabilization and loss of protection by F-actin at 1 M KCl. Thus, it was concluded that the different magnitude of the stabilization detected among fish species was determined by myosin not F-actin. At present, it is uncertain what differences in S-1 structure cause the different extent of stabilization. Regarding the species-specificity in myosin properties, the most prominent one is the habitat temperature dependent thermal denaturation rate of myosin.7 Species-specific filament forming ability has also been reported accompanying a characteristic actin-activated Mg2+-ATPase activity.26 In this paper, we added a new specificity, that thermal denaturation mode of myosin as studied by S-1 and rod denaturation is species-specific. The
Thermal denaturation pattern of fish myosin
FISHERIES SCIENCE
species-specific denaturation modes obtained in this paper are ones at 0.1 M KCl pH 7.5. In order to understand the role of myosin denaturation in the thermal gelation process, it is essential to study the denaturation mode in the presence of high salt such as 0.5 M salt, or in a dissolved form as actomyosin. These will be described in another paper. REFERENCES 1. Connell JJ. Studies on the proteins of fish skeletal muscle. 7. Denaturation and aggregation of cod myosin. Biochem. J. 1960; 75: 530–538. 2. Arai K, Takahashi H, Saito T. Studies on muscle protein of fish III. Inhibition by sorbitol and sucrose on the denaturation of carp actomyosin during frozen storage. Nippon Suisan Gakkaishi 1970; 36: 226–236. 3. Focant B, Huriaux F. Light chains of carp and pike skeletal muscle myosin. Isolation and characterization of the most anodic light chain on alkaline pH electrophoresis. FEBS Lett. 1976; 65: 16–19. 4. Kimura I, Murozuka T, Arai K. Comparative studies on biochemical properties of myosins from frozen muscles of marine fishes. Nippon Suisan Gakkaishi 1977; 43: 315–321. 5. Murozuka T, Takashi R, Arai K. Relative thermo-stabilities of Ca-ATPase of myosin and actomyosin from tilapia and rabbit. Nippon Suisan Gakkaishi 1976; 42: 57–63. 6. Murozuka T, Arai K. Purification and thermo-stability of myosin Ca-ATPase from the frozen muscle of yellowfin tuna. Nippon Suisan Gakkaishi 1976; 42: 65–70. 7. Johnston IA, Freason N, Golberg G. The effect of environmental temperture on the properties of myofibrillar adenosine triphosphatase from various species of fish. Biochem. J. 1973; 133: 735–738. 8. Hashimoto A, Kobayashi A, Arai K. Thermostability of fish myofibrillar Ca-ATPase and adaptation to enviromental temperature. Nippon Suisan Gakkaishi 1982; 48: 671–684. 9. Katoh N, Uchiyama H, Tsukamoto S, Arai K. A biochemical study of fish myofibrillar ATPase. Nippon Suisan Gakkaishi 1977; 43: 857–867. 10. King L, Lehrer SS. Thermal unfolding of myosin rod and light meromyosin. Circular dichroism and tryptophan fluorescence studies. Biochemistry 1989; 28: 3498–3502. 11. Kato S, Konno K. Isolation of carp myosin rod and its structural stability. Nippon Suisan Gakkaishi 1993; 59: 539– 544.
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