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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.

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.

Adenosinetriphosphatase (ATPase) activity of actomyosin of the obliquely striated mantle muscle of squid was studied to confirm and supplement the previous work. ATPase activity was assayed at ionic strength of 0.15 or 0.6, Tris-maleate buffer 30 mM (pH 7.0-7.5), protein 0.1mg/ml, ATP 3.2 mM, CaCl2, MgCl2 or EDTA 3.2 mM, and 25°C; for 10 min. Effects of Ca2+ and Mg2+ were tested in the function of temperature. Both Ca2+ and Mg2+ revealed marked activation at eithe ionic strength of 0.15 or 0.6, but EDTA none. At ionic strength 0.15, Ca2+-ATPase showed a steep peak at 30°C, while Mg2+-ATPase demonstrated a gradual peak with the top at 35°C. At ionic strenght of 0.6, the macimum of the former appeared at 25°C and that of the latter at 40°C. Mg2+ ion seemed to stabilize the ATPase. When the activety was assayed at various ratios of actomyosin and ATP, the full activity levels were attained below the ratio of 0.1-0.2 mg/ml protein per 3 mM ATP. Addition of Mg2+ to Ca2+-activated ATPase, reduced the activity to the level activated with Mg2+ alone, while addition of Ca2+ to Mg2+-activated ATPase, did not alter the activity. Effects of Mg2+ appeared to overcome that of Ca2+. During 60 min incubation of enzymic reaction mixture at 25°C prior to adding ATP, the activity was reduced appreciably. This seemed to satand with the so far reported lability of the squid actomyosin which was illustrated in changes of several physiconchemical properties.

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.

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) 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.

In the previous papery1), it was shown that on dehydration of muscle of flatfish or squid, “Myosins s”** were readily denatured -the extractability decreased-, while alubumins remained unchanged. The question, however, was left open whether the proteins extractable after dehydration are the same as before. In order to answer it, the changes in viscosity and streaming birefringence (SB) of extractable proteins during dehydration process were followed, using thin slices of flatfish muscle as material. Since it was found2) that in fish “Myosin”, different from rabbit “Myosins”, viscosity number, In ηr/c (ηr: relative viscosity, c: protein concentration), is not independent to c, viscosity was determined at two protein concentrations, one above and one below 0.7mg/ml, and the result was expressed in viscosity number at concentration 0.7 mg/ml. Each determination was carried out, using the tilting viscometer2), at velocity gradients 200 and 500 sec-1. Streaming birefringence was compared by minimum protein concentration which gives a distinct cross of isocline under crossed Nicols. Results. (i) Viscosity number of salt extractable proteins (L) decreased in parallel to rate of dehydration (Fig. 1, a, b). However, one cannot safely deduce from this the change in viscosity of “Myosins” in the course of dehydration, because “Myosins” content of this fraction vividly decreased on dehydration as shown in Table 2, and because in fish “Myosins” viscosity number lowers with decreasing concentration 2). (ii) Viscosity number of Fraction S2 prepared according to the scheme shown in Table 1, was also found to decrease with dehydration (Fig. 2, a and b). “Myosin” content of this fraction kept nearly constant up to 8 day dehydration (Tables 4 and 5). Therefore, one can safely conclude the decrease in viscosity of “Myosins” with dehydration of meat. (iii) Streaming birefringences of “Myosins” decreased in the course of dehydration of meat (Tables 3 and 6). It may be concluded that dehydration causes not only decrease of extractability of “Myosins”, but also changes in shape of the particles, such as decrease in asymmetry.

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) To prove the assumption that the streaming birefringence (SB) observed in the aqueous extracts of squid muscle is due to the dissolving-out of actomyosin or an actomyosin-like protein, the streaming birefringent protein was separated as a concentrated solution by precipitating it through dialysis against 0.3M KCl. Then the properties of the protein were compared experimentally with those of carp actomyosin. 2) Whereas SB of carp actomyosin, on addition of ATP, showed a very similar response to that reported on rabbit actomyosin (Fig. 2), the streaming birefringent protein of squid behaved itself in a similar but distinct way, if Mg ion was present (Fig. 3). 3) Both carp actomyosin and the streaming birefringent protein of squid showed a typical superprecipitation (Figs. 5 and 6, Tables 2 and 3). 4) From salting-in curves, it was found that the used preparation of the streaming birefringent protein of squid contained one or two minor components, while that of carp actomyosin was homogeneous (Figs. 7). 5) It was inferred that, in the aqueous extracts of squid muscle, an amount of actomyosin-like protein is dissolved which has the functional properties of the contractile proteins. However, for some differences from actomyosins of rabbit and carp were found, and for these differences may be shared by mollusks, it was proposed to call this protein “M-actomyosin”.

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.

The effect of two-step Heating, including low temperature setting, on the gel properties of the paramyosin-myosin system was investigated. At relatively low paramyosin contents, little effect of the two-step heating on gel properties was observed. When the paramyosin became 25 % or more, the indices of gel properties became significantly larger than those for the gels prepared by one-step heating. Paramyosin was more susceptible to the effect of two-step heating than myosin. It was thus concluded that paramyosin was mainly responsible for the effect of setting in invertebrate muscle gels.

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.