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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.

The cryoprotective effect of monosodium glutamate (MSG) and lysine hydrogenchloride (Lys-HCl) on lactate dehydrogenase was investigated in comparison with the effect of sodium chloride. The cryoprotective effects of MSG and Lys-HCl as solutes seem related to their zwitterionic properties. Thus their pH buffer action and physicochemical modifying actions on the freezing process of water were examined. No pH buffer action, as estimated from color changes of a universal pH indicator, was observed for either 0.2 M MSG or Lys-HCl. Physicochemical modifying actions, such as freezing-point depression and supercooling, were demonstrated on all the additive solutions. The pH buffer action of the additives was probably not related to cryoprotective effect, while the termination of the supercooling, more precisely the formation of ice, was found closely related.

Calmodulin (CaM) was purified from squid mantle muscle using hydrophobic chromatography. Relative molecular mass of the purified CaM deduced form the SDS-PAGE was exactly the same as that of bovine brain. Comparison of bovine brain and squid CaM indicates that each activates phosphodiesterase identically and that their activation curves are superimposable. Distribution of CaM in the squid tissues was also examined by measuring the activation level of PDE activity. The content of squid CaM in generative organ and fin is relatively higher than in pancreas, liver, and mantle muscle.

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.

Ultrafiltered fur seal muscle hydrolysate was divided into eleven fractions by gel filtration on Sephadex G-15. One of the fractions (Fraction G9) accelerated the ATPase activity of carp myosin B to a rate about two-fold faster than that of the control. Fraction G9 showed a single ninhydrin spot in its silica gel thin layer chromatograph, and gave a positive test for tryptophan by the p-dimethylaminobenzaldehyde method, while tests for tyrosine, and for arginine were negative. The ion exchange amino acid analysis of its acid hydrolysate showed a predominant content of lysine, nearly equivalent to the amount of tryptophan determined from its UV absorbancy and the p-dimethylaminobenzaldehyde method. The N-terminal amino acid analysis gave di-DNP-Lys as the sole DNP-amino acid. The structure of the ATPase accelerating peptide fraction, Fraction G9, was deduced to be Lys-Trp.

The object of this study is to estimate objectively the species difference and the changes in he physical properties of fish meat during the post-harvest storage and to search for the changes of the muscular matters underlying the property changes. Muscles of five fish species: skipjack, flyingfish, common horse mackerel, plaice, and channel rock fish were stored at 4°C for 14 days. Sample fish were taken out in turn and submitted to measurements of physical properties, namely penetration of a needle, firmness and cohesiveness by a General Foods type Texturometer, respectively. The measured values of each measurement item varied from species to species. During the storage, each of them shifted to the values reflecting the softening of the muscle usually observed. The rate of softening varied from species to species. The extractability of myofibrillar-, sarcoplasmic-, alkali-soluble-, and stroma-protein fractions of five fish muscles did not show any significant changes during storage. The SDS-polyacrylamide gel electrophoretic patterns of these extracted protein fractions and of the myofibrillar proteins extracted after M. H. STROMER showed no difference with respect to the storage time. The muscle samples were broken by rotating blades and sieved by 7 mesh, the percentage of the pieces remaining on the sieve decreased with the decrease of the freshness. The muscle samples were homogenized and the length of the myofibril fragments or the sarcomere number of each fragment was estimated under a microscope. The percentage of the small myofibrils varied by the fish species, and the degree of fragmentation increased with the storage period. These results denoted that changes in the physical properties, namely softening of meat, during the post harvest storage were affected more by the changes of the muscle tissue structures than by the changes of the component proteins.

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.

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.

1. 1. The acid cysteine proteinase was purified from the squid mantle muscle by affinity chromatography on a leupeptin-TOYOPEARL column. 2. 2. The molecular weight of the squid cysteine proteinase was estimated to be 21,000 by SDS-polyacrylamide gel electrophoresis and to be 26,000 by gel filtration chromatography. 3. 3. The activity of the squid cysteine proteinase was inhibited by inhibitors for cysteine proteinases, such as chymostatin, E-64, TPCK, TLCK and NEM. 4. 4. The squid cysteine proteinase hydrolyzed hemoglobin, hemocyanin, casein, myoglobin and bovine serum albumin.

The actomyosin ATPase of the obliquely striated squid mantle muscle, which has many characteristic properties in its solubility, electrophoretic behavior and lability as reported previously, was characterized. The ultracentrifugal patterns of squid actomyosin showed its peaks in aggregated states, while, on SDS-acrylamide gel electrophoresis, amount of actin and myosin accounted for 70% of total proteins. Paramyosin and tropomyosin were found as contaminants which are not readily removable at present. The ATPase activity measured in tris-maleate buffer at 0.05 or 0.6 ionic strength (I) and at 20°C was stimulated markedly with Ca2+ and moderately with Mg2+, but depressed with EDTA. [Stimulation by both Ca2+ and Mg2+ was most effective at 32 ram.] In the presence of Ca2+, the activity showed 2 maxima at pH 7.0-7.5 and pH 9.0, while, with Mg2+ present, it leveled off beyond pH 6.8-7.0. The activity was highest at I=0.25. The presence of membrane and mitochondrial ATPase was refuted since neither ouabain nor oligomycin depressed the activity. The stimulating effect of Mg2+ appears to he characteristic of the squid actomyosin ATPase.

This work was undertaken as an introductory step for isolating the biologically active components of the hydrolysate preparation of fur seal skeletal muscle proteins which contain some vasoactive factors as reported previously. This study was carried out with 2 preparations obtained at different fractionation stages, i.e., “unfractionated mixture” and “lyophilized powder”, the latter constituting the second fraction of gel filtration (Sephadex G-25) of the former. The 2 preparations differed in peptide composition, because their relative biuret, ninhydrin and ultraviolet absorption values differed respectively. By paperand thin layer-chromatography, peptide mapping as well as ion exchange column chromatography, both preparations were found to contain more than 22 peptide components, though the lyophilized powder had less than the other. The lyophilized powder showed an accelerating effect on the rates of both superprecipitaion (0.1 ionic strength, pH 7.0) and ATPase activity (0.15 ionic strength, pH 6.4) of fish actomyosins. The acceleration was also positive when Ca and Mg ions were removed by adding GEDTA or EDTA. These effects seem to be involved in the vasoactive and vasodilatory function of the fur seal peptide preparation.

The water distribution in the pulvinus of Mimosa can be visualized by an NMR imaging technique. After stimulation of a Mimosa plant, water in the lower half of the main pulvinus disappeared, the water previously contained in this area seeming to be transferred to the upper half of the main pulvinus. Movement of the water in conjunction with Mimosa movement was visualized sequentially by a non-invasive NMR imaging procedure.

1. 1. The profiles of the proteolytic activity in autolysis of the mantle muscle of the squid, Ommastrephes sloani pacificus , was studied by measurement of the released peptides and by SDS polyacrylamide gel electrophoresis. 2. 2. In the acidic ranges, there was found high proteolytic activity with a maximum at pH 3.1. This activity was ascribed to a cathepsin D-like proteinase. 3. 3. The activity above the neutral pH was much lower than that of the acid proteinases, and appeared to be due to metallo- and thiol-proteinases.

TNCs from lobster, mussel, and squid migrated with rabbit TNC at an apparent mol. wt of 18,000. Electrophoretic mobilities in the presence or absence of Ca2+ were compared: the electrophoretic mobility of rabbit TNC was greater in the presence of Ca2+ than it its absence, but all invertebrate TNCs tested migrated identically, whether Ca2+ was present or not. The Ca2+-binding capacity of invertebrate TNCs was only one Ca2+ ion per molecule. The alpha-helix contents in the presence or absence of Ca2+ were compared: rabbit TNC changed by a value of 16% and invertebrate TNCs by 4%. Antibodies to loligo TNC did not cross-react with rabbit TNC, but did interact with their molluscan TNCs.

1. 1. When the proteinases of the squid mantle muscle were extracted in the presence of dithiothreitol (DTT), the acid proteinase activity increased, indicating that the squid mantle muscle contains a considerable amount of the acid thiol proteinase. 2. 2. The crude extract hydrolyzed neither α -N- benzoyl- d , l -arginine-ϱ-nitroanilide (BAPA) nor azocasein, thus refuting the presence of cathepsins B and L in the mantle muscle. 3. 3. The cathepsin D-like proteinase and the acid thiol proteinase were separated by Sephadex A-50 column chromatography. 4. 4. Each of the above partially purified proteinases was able to degrade carp actomyosin at pH 2.5 and 5.0, respectively.

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.

In the previous paper, the ATPase accelerating peptides were found to be distinct from the vasoactive and vasodilatory peptides in the same hydrolysate. This work was undertaken to isolate the ATPase accelerating peptide from the fur seal muscle hydrolysate. Minced skeletal muscle of fur seal was digested with protease and the resulting hydrolysate was fractionated with 90%ethanol. The obtained precipitate and the effective agent was fund only in the precipitate. By continuous flow electrophoresis, the was divided into five major fractions, each of which was examind for accelerating effect on ATPase by two different methods, i.e., inorganic phosphate determination and pH-stat method. Among the five electrophoretic fractions, the second fraction E2 migrating to the anode showed the best accelerating effect, while the other fractions were less accelerative. Also the possible of Ca ions was refuted. E2 was also examined for physiological activity by kymographic recording of the contraction of rat intestine smooth muscle and showed an effect which is revealed by a contraction preceded by a slight relaxation. Thin layer chromatography revealed that there are more than two peptide components in fraction E2.

1. 1. A novel lectin (Achatinin) was isolated from the body-surface mucus of African giant snail, Achatina fulica Ferussac. 2. 2. The lectin recognizes N-acetylneuraminic acid preferentially and also other N-acetyl aminosugars to some extent, while it does not bind to N-glycolylneuraminic acid. 3. 3. The molecular weight of the lectin estimated by SDS polyacrylamide gel electrophoresis was 78,000. 4. 4. The lectin requires Ca2+ for its haemagglutinating activity. 5. 5. The isolated lectin was free of antibacterial activity which has been detected in the body-surface mucus.

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.

Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied. LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C. The speed of the inactivation differed in each. The stability of the enzymes increased with the increase of the enzyme concentration. Sodium glutamate and albumin prevented the freeze inactivation. While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly. The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively. Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks. The enzymes tested lost some part of their activity when their solutions were diluted by the media. This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media.

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) The distribution pattern of squid muscle proteins was studied by the method of DYER et al. as well as by a similar method which was modified in some points so as to fit the properties of squid muscle proteins. 2) Squid muscle proteins were found to consist of 12 ?? 20% of non-myosins, 77 ?? 85% of myosins and 2 ?? 3% of stroma. Subdivision of non-myosins into myogen, myoalbumin and globulin X did not give a uniform pattern from analysis to analysis. As far as the gross distribution pattern of muscle proteins is concerned, squid seems to resemble fishes, although properties of its myosins are distinct from those of fishes as shown in the previous reports.

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 Amarked streaming-birefringence was found in the aqueous extract of the arm muscle of the squid, while none in that of the mantle muscle. 2 On electrophoretic analysis, both arm and mantle extracts showed patterns consisting of 3 components. However, the values of the electrophoretic mobility suggest that the slower two components of the arms correspond to the faster two of the mantle, and that the fastest component of the arms is responsible for the streaming-birefringence. 3 The mobility values of the components were found to increase slightly after frozen storage. 4 The nature of the streaming-birefringent protein has been discussed, existence of a protein but myosin or actomyosin being suggested.

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. 1. The ATPase of Mimosa pudica pulvinus hydrolyzed various nucleoside triphosphates, but the presence of divalent cations was required for obtaining positive activity. 2. 2. The enzyme also hydrolyzed the nucleoside diphosphates but it did not hydrolyze AMP, PPi or PNPP. 3. 3. On the basis of its activity with both triphosphates and diphosphates, quantitative relationships between ATP, ADP and AMP were traced by HPLC. 4. 4. The present enzyme did not hydrolyze ATP and ADP at the same time, but it first hydrolyzed the ADP rapidly and then the ATP rather slowly. 5. 5. The ATPase isolated from Mimosa pudica pulvinus is an ATP-ADPase of a type which differs from the known enzymes such as apyrase.

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.

Lactate dehydrogenase (LDH) was chemically ethyl-acetimidated (EA-), dimethyladipimidated (DMA-), carbamylated, acetylated, acetoacetylated, or succinylated in order to alter the ionic charges on the epsilon-amino group of lysine residues. Acetylation, acetoacetylation, and succinylation, which change the positive charge at the lysine side chains to a negative one, inactivated the enzymic activity, but the rest of the modifications exerted no such inactivating effects. The active modified enzymes were subjected to freeze denaturation study, using the enzymic activity as an indication of the degree of the denaturation. The active enzymes were diluted with deionized water and stored in a freezer (-23 degrees C) for 1-3 days. Enzymic activity was assayed immediately after thawing. All the modified enzymes retained their activity even after the 3-day frozen storage, while the control or native enzyme lost its activity within 1 day of storage. Furthermore, the modified LDHs freeze-stored in 0.2 M monosodium glutamate (MSG) or 0.2 M lysine-hydrochloride (Lys-HCl) retained their activity. The cryoprotective effects exerted by the modifications and by 0.2 M MSG seemed to be synergistic, whereas those exerted by the modifications and by 0.2 M Lys-HCl did not. The mechanisms of cryoprotection and freeze denaturation are discussed in relationship with the cryoprotective effect exerted by already known cryoprotectants, such as sucrose or dimethyl sulfoxide.