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Structural changes of actomyosin, the major protein of muscle, on heating have been estimated on ATPase activity. We investigated carp actomyosin molecule changes on heating based on biophysical and biochemical techniques. Actomyosin molecules began to unfold at ∼30°C. Hydrophobic amino acid residues and SH groups, which had been inside the molecule, emerged to the surface. Because of hydrophobic interactions and disulfide bonds, actomyosin molecules formed aggregates. At > 40°C, a part of myosin molecules was dissociated from actin filaments. Thus, dissociated myosin and the myosin-lacking molecules co-existed. In addition, fragmentation of actin filaments was observed, which was associated with the dissociation of myosin molecules. At ≥ 60 °C actomyosin molecules formed larger aggregates, in which no filamentous shape was observed. This aggregation occurred mainly by formation of SS bonds.

To elucidate the roles of the head and the tail portions of the molecule in the thermal gelation of myosin, the gelation characteristics of heavy meromyosin (HMM) and of light meromyosin (LMM) were investigated. The aggregation process of HMM corresponded to that of myosin alone in the temperature range above 50°C. Both the dynamic viscoelastic behavior and the aggregation process of LMM agreed fairly well with those of myosin alone in the temperature range up to 45 °C. Therefore, the first development of gel elasticity of myosin was attributable mainly to the tail portion of the molecule and the second was to its head portion.

The effect of ionic strength on the thermal gelation process of myosin was investigated by dynamic viscoelasticity measurements. The dynamic viscoelastic behavior of myosin was divided into three ionic strength groups. Each ionic strength group was closely related to the state of myosin molecules before the rise in temperature. Both the head and the tail portions of the molecule participated in the gel formation of myosin, but the temperature ranges differed. It was proposed that the first development of gel elasticity of myosin (30–45 °C) was attributed many to the tail portions of the molecules and that the second development (above 50 °C) was mainly to the head portions.

Isolation of myosin from the obliquely striated muscle of squid mantle was studied by comparing the various conventional isolation methods used for the vertebrate and invertebrate muscles. A method suitable to the squid myosin is proposed. The purity of each obtained preparation was checked by SDS polyacrylamide gel electrophoresis. The myosin preparations obtained by the conventional methods did not give any band corresponding to the myosin heavy chain, but those less than 14×104 in molecular weight were not free from contaminating proteins such as paramyosin and actin. The myosin preparation obtained by the proposed method showed an electrophoretic profile with a heavy chain band (18-20×104) and two light chain bands (1.2 and 1.5×104) of myosin with a faint band of paramyosin (10×104) in 7.5% gel. The tryptic digestion of the squid myosin shows that it is less resistant than fish myosins, and much less than rabbit myosin. The difficulties in obtaining an intact heavy chain during the isolation procedures were attributed to the fragility of the squid myosin molecule and to the possibility of high catheptic activity in the squid muscle.

To determine the contribution of tropomyosin to fish muscle gel characteristics, the gel properties of the tropomyosin-desensitized actomyosin system and of the tropomyosin-myosin system were investigated. For both systems, the indices of gel properties decreased considerably on increasing the tropomyosin content. Even when the two-step heating was carried out, the indices of gel properties decreased considerably on increasing the tropomyosin content. Tropomyosin negatively affected the gel formation of fish muscle and reduced the gel strength and elasticity of the fish muscle gels.

Tropomyosin preparations from obliquely striated mantle muscle of squid were obtained from low ionic strength extracts of natural actomyosin and their physico-chemical properties were studied. The sedimentation coefficient, 3.0 S, and the intrinsic viscosity, 0.31dl/g, of squid tropomyosin were very similar to those of skeletal tropomyosin. The relative viscosity decreased with increasing ionic strength, becoming constant over 0.1 M KCl. The molecular weight of the squid tropomyosin subunits, 3.5 × and 3.7 × 104, obtained by SDS disc electrophoresis was slightly higher than those of other species. There are some differences between squid and vertebrates in the contents of some amino acids, such as Pro, Trp and Arg. Moreover, the squid tropomyosin was poorer than the skeletal and the non-muscle tropomyosins in easily forming the paracrystals and did not form any crystals.

Denaturation of squid actomyosin during frozen storage was studied by measuring solubility, viscosity, and ATPase activity, and by ultracentrifugal analysis and SDS-electrophoresis. The additive effect of sodium glutamate, well-known for its cryoprotective effect on the freeze denaturation of carp actomyosin, was also checked. When solutions (0.6 M KCI) or suspensions (0.05 M KCI) of the isolated squid actomyosin were stored at -20 ??, the solubility, reduced viscosity and ATPase activity decreased with the length of frozen storage. The ultracentrifugal patterns showed that aggregation proceeded during frozen storage. Evidently, sodium glutamate prevented the freeze denaturation of squid actomyosin, as in the case of carp actomyosin. When the mantle muscle of squid was freeze-stored at -20 ?? and extracted with 0.6 M KCI, the amount of soluble actomyosin extractable from the frozen meat and the ATPase activity of the actomyosin were decreased only slightly even after a long freezing period. This differed from the results with isolated actomyosin.

G-Actin prepared from the obliquely striated mantle muscle of squid by a modified SPUDICH and WATT's method was ultracentrifugally monodisperse with a sedimentation coefficient, S020, w, of 3.3S, and its molecular weight was estimated to be 4.3×104 by SDS disc electrophoresis. The intrinsic viscosity of the squid G-actin was 0.12dl/g, and the viscosity increased by adding 0.1M KCl or 2mM MgCl2 revealing polymerization to F-actin. On ultracentrifugation, the squid F-actin showed 2 faster peaks with sedimentation coefficients corresponding to those of rabbit F-actin. Under the electron microscope, squid F-actin consisted of double stranded filaments, on which a periodicity of 35-36nm was recognized as was also the case with rabbit F-actin. There were no significant differences in amino acid composition among the actins of squid, fish, rabbit and plasmodium. Superprecipitation and viscosity response with added ATP were tested with positive results on the synthetic actomyosin from squid actin and carp myosin. The latter test was compared with one on the natural actomyosin of squid. Squid actin was found similar to the actins of rabbit, fish, and plasmodium actins in the ability to interact with vertebrate myosin. In contraction of the obliquely striated mantle muscle of squid, squid actin appears to be functionally similar to that of vertebrate striated muscle.

The nature and mechanism of the freeze denaturation of fish myosin was clarified by measuring changes in solubility, ATPase activity, and filament reconstituting capacity over 8 weeks of storage at −20 °C. Carp myosin was stored in three states: (i) solution in 0.6 M KCl, (ii) dumbbell-shaped filament suspension, and (iii) spindle-shaped filament suspension in 0.05 M KCl. Each group was stored either in the absence or presence of 0.2 M sodium glutamate. Filament reconstituting capacity was estimated by examining electron micrographs of shapes obtained by definitive filament reconstituting procedures for either dumbbells or spindles. Without sodium glutamate added, a decrease in solubility, ATPase activity, and filament forming capacity indicated denaturation taking place. A rise and a rapid decrease of ATPase activity were noted at the early stage of storage. When sodium glutamate was present during the storage, the above changes were replaced by an outstanding increase followed by leveling off near or above the original value. These indicated the prevention of denaturation by sodium glutamate. Myosin appeared less resistant to freeze denaturation in a dissolved state than in a filament state.

To obtain basic knowledge for processing of the meat products, such as, kamabokos, sausages, and hams, the myofibrillar proteins of rabbit, carp, and scallop were isolated and submitted to the differential scanning calorimetry (DSC). Myosin preparations gave broadly dispersed endothermic regions in each of which a peak was found at 62°C for rabbit(R), 50°C for carp(C), and 40°C for scallop(S). LMM's gave a single peak at 60°C(R)and 47°C(C). The peaks of myosins reflected the denaturation of the LMM portion of the myosin molecule. Actins showed a peak at 82°C(R), 75°C(C), and 77°C(S). In the crude actin, the actin's peak was accompanied by a peak at 58°C(R), 53°C(C), or 42°C(S), each of which was attributed to the regulatory proteins. The natural actomyosin preparations illustrated 2 peaks at 49°C and 62°C for rabbit and 41°C and 52°C for scallop, but that of carp gave 3 peaks at 43°C, 53°C, and 68°C. Each second peak was ascribed to myosin. None of the rest were found to be correlatable with any of the actin, myosin, or the regulatory proteins and thus these peaks were assigned to some conjugate derivatives of actin and myosin. The peak top temperatures, i.e., the heat denaturation temperatures, of the respective proteins were found to be different for the different animal species and an order of “rabbit, carp, and scallop” was commonly observed with a little exception in the case with scallop actin. The former descreasing order was related to the order of the habitat temperatures of the tested animals.

Dispersion profiles of proteins in “muen” (non-salt) and “kaen” (salt added) frozen surimi (fish mince) were examined under electron microscope. The cross sections of the muen surimis gave various profiles but no appreciable difference related with the jelly strength of the drived kamabokos was found. The dispersion profiles of the muen surimis were classfied roughly into following four types: (1) a profile demonstrating well reserved myofibrillar structures in which the M-lines and the Z-bands remain in nearly intact state, (2) a profile demonstrating moyfibrillar structures in which the M-lines are inappreciable and the vague Z-bands are shown, (3) a profile demonstrating parallel streams of fine filaments which are probably assingnable as actomyosin filaments, (4) a profile in which no uniform structure is appeciable. The dispersion profiles in the kaen surimis were similar to those found in kamabokos (cooked jellies) and showed dense or loose matrices of cross-linked filaments. Dilute homogenate suspensions of the surimis illustrated two types of filaments which were probably assignable as actomyosin filaments and as naked F-action filaments beside aggregates of myosin. The grade SA surimis and some grade C surimis demonstrated fine arrowheaded filaments of actomyosin and gave good jellies. Other grade C surimis demonstrated filaments of myosin-poor actomyosin filaments and gave poor jellies.

The thermal gelation processes of the myosin-natural actomyosin system were investigated to determine the role of F-actin in thermal gelation of actomyosin. The dynamic viscoelastic behavior during thermal gelation changed considerably depending on the (F-actin)/ (myosin) ratio. F-actin gave the viscosity to an actomyosin sol but did not affect the elasticity development occurring in the 30 - 46°C range. The decrease in storage modulus in the 46 - 53°C range was directly induced by the presence of F-actin. The gel of myosin alone showed the highest elasticity, while that of myosin containing a small amount of F-actin had the highest elastic modulus.

Even when actomyosin dissociated into myosin and actin in the paramyosin-natural actomyosin system, the indices of gel properties increased on increasing the paramyosin content; however, their increases were smaller. Also, in the paramyosin-myosin system gels, the indices of gel properties increased on increasing the paramyosin content; however, the increases were smaller than those for the paramyosin-natural actomyosin system gels. In both systems, a high linearity was observed between each index of gel properties and paramyosin. It was likely that the paramyosin-myosin interaction did not bring about the characteristic texture of invertebrate muscle gel directly, but the binding of myosin and actin might be indispensable if the contribution of paramyosin was to occur.

1) Some electrophoretic study was made on M-actomyosin, an actomyosin-like protein obtained from aqueous extract of the squid muscle. The actomyosin-like pre-paration showed an electrophoretic diagram including two or three peaks, which is similar to that of the actomyosin-like protein from salt extract of the squid muscle. Also in respect of the electrophoretic mobility each component corresponded with each other in both cases. So the two actomyosin-like protein, the one from aqueous extract and another from salt extract of the squid muscle, including the accompanying proteins, were found to be identical. There was found an indication that Component 2, the middle-speeded is rather scanty in the case with the protein from aqueous extract. 2) When a precipitate of the actomyosin-like protein from salt-extract was extracted repeatedly with water, a better part of the protein moved into solution. Moreover, marked streaming birefringence (SB) was found in the extracts as the case with the repeated aqueous extraction of the squid muscle. On the contrary, SB was never found in the case with carp actomyosin, whereas nearly half of the used actomyosin of the carp was brought in solution. 3) From these experimental bases, it was proposed that the actomyosin-like protein which occupies the dominant part of the squid muscle proteins should be referred to as “M-actomyosin” no matter whether it is isolated from salt extract or from aqueous extract.

1) As far as the salting-in curve is concerned, it appeared that 2 or 3 components are contained in M-actomyosin preparation obtained by adding KCl to the streaming birefringent aqueous extract of squid muscle. Among these components, Component I which is least soluble in lower ionic strengths (I) was found to be responsible for the characteristic responses to ATP and thus was inferred to be M-actomyosin. 2) Observations were made and discussed on the purification of Component I by reprecipitation. Thus it was inferred that reprecipitation is useful for purification, but that it is questionable if the purification was complete or not. It was also found that Component I is considerably unstable and is apt to change its solubility behaviors which is not the case with rabbit and carp actomyosins. 3) The salting-in range of M-actomyosin of squid was found to cover I = 0.2 ?? 0.5. In this respect, it appeared not very different from rabbit or carp actomyosins.

1) On M-actomyosin preparation of squid muscle purified by reprecipitations, its responses of streaming birefringence (SB) and of viscosity to adenosinetriphosphate (ATP) added were studied togather with superprecipitation. 2) The changes of SB stood in harmony with those of viscosity, suggesting that dissociation and recombination of M-actomyosin occurred successively when ATP was added. Superprecipitation was also confirmatively found with M-actomyosin. 3) In the process of ATP-responses of SB and viscosity, the adenosinetriphosphatase (ATPase) activity of M-actomyosin was found to be inhibited by Mg ion. It was shown that, as far as the effect of alkali earth metals on ATPase activity is concerned, M-actomyosin of squid is not distinct from actomyosin of rabbit and carp.

1. With carp myosin B ATPase, the acceleration effect was found at peptide concentration ranging between 1 nM and 1 mM with a maximum at 10 nM (30% acceleration) at the low myosin B concentration (0.2 mg/ml). At the high myosin B concentration (0.8 mg/ml) a maximum was observed at 1-10 mM (10% acceleration). 2. With rabbit myosin B ATPase, the acceleration (10%) was found only at a microM of Lys-Trp in the case of low myosin B concentration (0.2 mg/ml). Under the high concentration of rabbit myosin B, a maximum acceleration (150%) was found at 5 mM of the synthetic Lys-Trp. 3. Effect on superprecipitation was tested only at the high concentration of rabbit myosin B (0.8 mg/ml). The acceleration increased with increasing concentration of Lys-Trp up to 10 mM.

Ca2+-sensitivity of squid muscle proteins was studied by competition tests between squid actin and carp myosin on squid myosin B and by cross combination tests between squid myosin and actin and between carp myosin and squid thin filament fraction. The results suggest that the regulatory system of the squid mantle muscle is actin-linked as in skeletal muscle rather than myosin-linked. This view is supported by the presence of troponin-like components on SDS-disc electrophoresis of squid thin filament fraction.

1) Denaturation of carp actomyosin during storage at -20 degrees was studied with particular interest in the cryoprotective effect of sodium glutamate, the most cryoprotective of the compounds tested previously. 2) Storage with glutamate prevented the rapid decrease in solubility, viscosity, and ATPase (EC 3.6.1.3)activity of actomyosin during storage. Ultracentrifugal studies suggested that aggregation occurred in the frozen state without glutamate, but that added glutamate prevented aggregation or denaturation. 3) Electron microscopy showed that the original actomyosin consisted of long filaments with typical "arrowhead" structures, and that these decomposed into small fragments and sticked with globular portions, forming loosely packed aggregates during storage without glutamate. On storage with glutamate, the filaments were well preserved, and their fine structure was clearer than that of the original sample. 4) Preparations of actomyosin extracted with 10 mM glutamate were of better quality and their ultrastructure and physicochemical and biochemical properties showed increased stability on freezing. 5) Freeze-denaturation seems to involve complicated aggregation with transconformation of proteins besides the side-to-side aggregation discussed previously.

Although a relatively large amount of actin is contained in myofibril, the role of F-actin in the thermal gelation of muscle proteins has been little studied. We investigated the dynamic viscoelastic behavior of F-actin on heating to clarify the role of F-actin in the thermal gelation. F-actin did not form an elastic gel on heating but did turn into a curdy matter. In addition, F-actin showed no development either of the storage modulus or the loss modulus during a rise in temperature. These results suggest that F-actin negatively affects the gel formation of fish muscle proteins.