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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.

1. The morphology of bacteria treated with the bactericidal glycoprotein, Achacin, purified from the giant African snail, Achatina fulica Férussac, has been studied. 2. Achacin lengthens the bodies of Escherichia coli by three to seven times. 3. Achacin damages the surface of Staphylococcus aureus and sinks the cytoplasmic membranes into the cytoplasm. 4. Achacin causes neither the leakage nor the destruction of cells.

1. Bactericidal action of a glycoprotein, Achacin, purified from the giant African snail, Achatina fulica Férussac, has been studied. 2. Achacin kills both gram-positive and gram-negative bacteria, but only in their growing states. 3. Achacin does not have any bacteriolytic activity. 4. The strain which has no cell wall is a little more sensitive than the native strain and the cell membrane-damaged strain. 5. Achacin was observed on the cytoplasmic membrane and on the cell wall of treated Escherichia coli by immunoelectron microscopy. 6. Achacin attacks the cytoplasmic membrane of the cell.

Mackerel water-soluble protein concentrate (SPC), which had been concentrated by an ultrafiltration system, were treated with several polymer coagulants to recover protein at high concentration. Over 90% of the protein in the SPC was aggregated by coagulating agents. The aggregation ratio depended on the pH of the reaction. The highest ratio of aggregation was obtained at about pH 5.0. In addition, the effects of alcohols and organic acid salts in aggregating the protein in the SPC were studied. The ratio of the aggregation increased slightly by the addition of alcohols, but not by the addition of organic acid salts.

イカ肉を加熱後,なま粕に10%の清酒および15%の焼酎, 16.5%の砂糖を添加し,食塩濃度が3,7%, 5.0%, 7.2%になるように食塩を加えた調製酒粕3種中に10日間保存して,その物性とタンパク質の変化を検討し,次の結果を得た.(1) 食塩3.7%調製粕を用いた粕漬処理肉の官能検査では, 2, 6, 10日間の保存で, 10日後の肉が,最もやわらかく,かみ切りやすく,弾力がなく,総合的に好まれた.さらに, 10日後の粕漬肉と加熱酒粕を用いて処理した肉とを官能検査によって比較すると,加熱粕漬肉の方が粕漬肉よりもかみ切りにくく,弾力が強く,総合的に好まれなかった.これらの評価より,粕漬肉のテクスチャー変化が保存だけの影響ではなく,酒粕中の酵素の作用によるものであることが示唆された.(2) テクスチュロメーターによる硬さの測定では,表皮の第1層,第2層のみを除いたイカ肉Iの粕漬肉は,漬け込み6日後までに硬さの値は急激に低下し,その後10日まで徐々に低下した.この結果は,官能検査結果に対応していた.表皮の第1層～第4層を除いたイカ肉IIの硬さはイカ肉Iよりも保存初期には低く,かつ低下の程度もイカ肉Iよりもゆるやかであることから,イカの皮は主に保存初期に,イカ肉は保存期間中を通じて粕の作用を受けるのではないかと考えられた.(3) イカ肉Iの引張強度は,粕漬処理により,縦方向(体軸方向)と横方向(体軸と直角方向)ともに低下し,特に横方向の強度低下が大きかった.イカ表皮の第3層,第4層を酒粕抽出液に浸漬すると,浸漬初期に縦方向の引張強度の低下が大きかった.(4) イカ肉は粕漬処理中にタンパク質が変化していることがSDS-PAGE分析により確認された.イカ肉の筋繊維タンパク質のミオシン重鎖,パラミオシン,アクチン,ミオチン軽鎖が減少しているのがみられた.また,イカ皮のタンパク質の低分子量化もみられた.以上より,イカ肉を粕漬処理したとき,テクスチャーの変化が認められ,肉,皮ともに物性が変化した.これは酒粕の中に含まれる酵素によりタンパク質の一部が分解することと関連があることが考えられた.

Cooked muscle tissue of five fish species were observed by optical and scanning electron micrography. Species with firm texture had thin muscle fibers with considerable heat-coagulating material between them; species having soft texture had thick muscle fibers with little heat-coagulating material. When the cooked muscles were compressed by a Texturometer, muscle fibers of the species having firm texture slid or shifted over one another to a lesser extent than those of species with soft texture. The heat-coagulating material seemed to obstruct the displacement of the fibers. The diameter and mobility of muscle fibers are determinative of firmness of fish muscle tissue.

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.

Mackerel water-soluble proteins' concentrates (SPC), which had been concentrated by the ultrafiltration system, were treated with three kinds of methods. The gel formation properties were also studied on the insolubilized SPC. Over 95% of protein in the SPC was insolubilized by two pH shifting methods: One was to adjust the primary pH of SPC lower than 3 then to 7, the other was to adjust the primary pH of SPC higher than 12 then to 7. The preferable gel was obtained by the pH shifting from 12 to 6. An elastic gel like Kamaboko was obtained from SPC by simple heating when SPC was highly concentrated (over 100mg/1). The alcohols caused insolubilization of SPC and the rate of the insolubilization increased with increasing of the carbon chain length of added alcohols. Insolubilized SPC with 50% ethanol formed a gel by heating, but its gelation property was poor. Fish water-soluble proteins were presumed not to cause gelation; we found, however, that the fish water-soluble proteins could form a gel if they were concentrated by an appropriate method like ultrafiltration.

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.

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.

This study was undertaken to indentify the key protein (s) which bring about the gel properties of kamaboko and other cooked gel products made from muscular proteins. Mixtures of rabbit actin and myosin of various ratios were prepared. To each mixture was added 2.5% NaCL and cooked in three differet ways: Method I, cooked at 85°C for 30min; Method II, held at 30°C for 30min and cookde as I; Method III, held at 4°C for 24h and cooded as I. Gels optained were submitted to the jelly strength measurement and the differential scanning calorimetry (DSC). The jelly strength values of the gels cooked by Method I increased with increasing myosin content. This indicated that myosin is the essential factor for high jelly strength. Effects of setting treatments of Method III in enhancing the jelly strength were more evidently manifested in the gels with higher myosin content. No effect was found with Method II. The R105 values of DSC which represent the water holding capacity of the gels paralleled the above jelly trength values, indicating that the highly hydrative property of myosin is an essential factor for the high jelly strength. Co-presence of actin and myosin was found essential to bestowing the precooking material with pastiness and consistency with pastiness and consistency which enable the precooking handling like molding or shape giving.

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.

The changes in viscoelasticity of natural actomyosin and myosin during thermal gelation were investigated by dynamic viscoelasticity measurements. Thermal gelation of natural actomyosin could be divided into four characteristic temperature ranges. The storage modulus increased considerably in the 32–43°C range, decreased sharply in the 43–52°C range, and then increased again in the 52–80°C range. For the thermal gelation of myosin, the storage modulus increased in two steps at two temperature ranges, i.e., 30–41°C and 51–80°C. An increase in the loss modulus was observed at an early stage of each of the two ranges.

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.

Contribution of the connective tissue content on the meat texture of fish species was studied. Firmness of texturometer tests and collagen content of the raw and cooked dorsal muscles of five fish species were determined. Five species selected were skipjack, flying fish, common horse mackerel, plaice and channel rock fish. The firmness value of raw meat varied in a decreasing order: plaice, channel rock fish, flying fish, skipjack, and common horse mackerel. That of cooked meat varied in a decreasing order: skipjack, flying fish, common horse mackerel, plaiceand channel rock fish. The order of the above series was in reverse between raw meat and cooked meat. The species with softer raw meat textures, on cooking gave a firmer texture. The amount of connective tissues as expressed by collagen content was determined on the muscle portion as the same with the texture measurements. Higher the collagen content, firmer the raw meat. There was a significant correlation (r: 0.697) between the collagen content and the firmness. The species with firmer raw meat texture contained higher collagen content than the species with softer texture. The amount of water soluble collagen at 20°C pH 7.0, assumed as an index of the exuding collagen during mustication, increased with the decrease of the firmness of raw meat (r: 0.650). The amount of water insoluble collagen gave higher correlation coe-fficient(r: 0.744) with the firmness. These results suggest that the properties and/or composition of collagen is varied by spies. However, between the cooked meat and the collagen content, such relationships was hardly recognized, and this was attributed to the collagen which is solubilized during cooking. There was no appreciable correlation between the amount of water soluble collagen at 70°C and the fumness of cooked meat, though cooked beef muscle has been reported to give such correlation. The connective tissue proteins must contribute to the raw meat texture, while the cooked meat texture does not depend upon the connective tissue proteins, but, probably on the other factors, like the muscle fiber characteristics as reported before.

Actomyosin solution isolated from carp muscle was frozen and stored with 20 amino acids. The cryoprotective effect of the additives was studied by following the changes in the solubility and the electron microscopic pictures of the protein. The results with the solubility change were classified into six groups with respect to the rate of the change; those with the electron microscopic pictures were classified into three groups depend-ing on the extent of the deformation of the filaments. Glu, Asp, and some others showed good cryoprotective effects in both the solubility and the electron microscopic pictures. Phe, Leu, and some others were less effective. Changes in solubility and in electron microscopic pictures were generally found to proceed in parallel to each other, but some exceptional cases were found for Pro, Gly, etc.

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.

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 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.

We designed a new method to evaluate the gel properties of fish meat gel products, by a stretching test, and a film-shaped gel sample, ca. 1mm thick, is used as a test piece. This method had the following advantages: The method can be applied to a small amount of sample and less than 500mg of sample was needed for one measurement. It had high accuracy and reproducibility. It showed high correlations with the pushing-in method using cylindrical test pieces, and values could be converted to those from each method. The fracture test and measurement of Young's modulus (non-fracture test) can be performed at the same time, while for the pushing-in method using cylindrical gels, the fracture test and measurement of Young's modulus must be done sep-arately. Thus the present stretching method was effective particularly when a large amount of sample could not be obtained, and also possessed several other advantages as above. Therefore, the present method may contribute for evaluation of the gel properties of fish meat gel products.

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.

In order to find suitable conditions for the fixation of fish muscles for electron microscopy, effect of the fixative concentration to the musculature of the freshwater and saltwater fish was examined. Carp and horse mackerel were used as the sample of the freshwate and saltwater fish, respectively. Muscle samples were prefixed in 3% glutaraldehyde-sodium cacodylate buffer (pH 7.2). Following the postfixation in 1% OSO4-sodium cacodylate buffer, muscles were dehydraed in ethanol and embedded in TAAB 812. The molarity of sodium cacodylate buffer was set to 0.05M, 0.1M or 0.2M for the freshwater fish, and to 0.1M, 0.2M or 0.3M for the saltwater fish, in both prefixation and postfixation Muscle samples were block-stained in 1% uranyl acette solution ater postfixation. Ultrathin sections were examined under electron microscope. The results show that the most suitable concentration of the fixatives is 0.1M and 0.2M for the freshwater and for the saltwater fish muscles, respectively. Use of the fixatives lower than the above strengths induced expansion of the mitochondria or the sarcoplasmic reticulum and broadening of the myofibrils in both fresh and salt water fish muscles. On the other hand, use of the fixatives stronger than the above induced deformation of the mitochondria, and obscuration of the filament structures.

Kasuzuke is a processing where fish meat and vegetables are cured in lees of sake (Japanese rice wine) for the purpose of preservation. In kasuzuke the fish meat cured in sake lees acquires a peculiar texture. Changes in muscle proteins were examined in relation with the texture changes. Spanish mackerel fillets were cured in sake lees after salting and were stored at 4°C for 1, 7, 14, 21 and 28 days. The fillets were submitted to the tests and measurements for firmness (after being cooked), extractability of proteins, viscosity and natural fluorescence of actomyosin solutions and the analysis of proteins by SDS-PAGE after various periods. For control, fillets were stored with the addition of 2% NaC1 or without any treatment. Firmness of the texturometer test increased after a day and 7 days, then decreased after 14 days and 21 days. Extractability of actomyosin and water soluble proteins did not change appreciably for 28 days. The changes in reduced viscosity and relative fluorescence intensity suggested that the conformations of actomyosin of muscle cured in sake lees have changed. SDS-PAGE patterns showed that the proteins of muscle cured in lees were decomposed during storage into fragments with smaller molecular weights. The decomposition of proteins were attributed to the effect of the acid proteinases in the sake lees. The texture changes during the curing in lees were suggested to come from the changes of proteins.

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.

With an aim of studying the gel properties and its formation, differential scanning calorimetry (DSC) was applied to the fish meat gels (kamaboko) in parallel with the jelly strength measurements. DSC curves of the gels were characterized by a single deep hollow with a bottom slightly below 100°C which represents the evaporation of water. Each curve was divided into two fractions, below and above 105°C (Q1 and Q2), and a quotient, R105 (=(Q2/(Q1+Q2))×100), was adopted to represent the amount of captured water in the total water of the gels. Two series of gels were prepared from Alaska pollack frozen mince, one with various protein concentrations, and another containing various amounts of egg white. The gels were submitted to DSC and jelly strength measuremetns. In both cases, R105 values illustrated good correlationships with the jelly strength values. That the state of water in the gels affects the gel properties like the jelly strength has suggested.

The goal of this study is to obtain a basis for the research of recovering and reutilizing the water soluble proteins discarded into the waste wash waters of the frozen mince (surimi) plants.Effect of an ultrafiltration system (IHI Co., Ltd.) to recover the water soluble proteins was studied. Proteins from 5 fish species, cod, Alaska pollack, sardine, mackerel, and horse mackerel were studied. The water soluble proteins were extacted with 5-10 volumes of water and the extracts were applied to the ultrafiltration system giving rise to concentrates. The protein concentration of the extracts, 0.1-2%, was raised to 0.4-18% with yields of nearly 90%. The low molecular matters, inorganic and organic, were largely filtered off by a membrane. The molecular weight composition of the water extracts and the concentrates were analyzed by Sephadex G-100 or G-200 gel filtration. The water extracts consisted of 5-7 groups with molecular weight ranging 4, 000-150, 000 and the proteins higher than 10, 000 were recovered in the concentrates by ultrafiltration with sieve size 20, 000. Some packing effect on the sieve was suggested. These profiles were generally found for the five species tested. The applicability of the ultrafiltration system appeared optimistic.

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 rheological properties of fish muscles of five species were characterized in terms of the creep compliance. A slight increase of the strain with time was observed in all species even without any load applied. The size of the linear region, where the stress increases in proportion to the strain, decreased when the loading time was prolonged. After 300s loading, the largest strain value of the linear region for each fish was, 9.5% in channel rock fish, 9.0% in plaice, 8.6% in flying fish, 8.5% in common horse mackerel, and lower than 2% in skipjack, respectively. On analysis of the creep compliance curves, a six-element model was found to represent each fish muscle, while three more elements, two ratchets and a spring, were needed to fit the creep recovery curves. The instantaneous modulus values of the creep compliance of flying fish and skipjack were larger than those of plaice and channel rock fish. Common horse mackerel gave an intermediate value. With the permanent viscosity modulus, plaice gave the largest value followed by flying fish, channel rock fish, common horse mackerel and then skipjack in a decreasing order. These results appeared to differ from the series obtained on our eating experience. It was suggested that our mouthfeel about “firmness” is not based on the difference in the elastic modulus of the muscles but on the ease of biting which is related to the collagen content as proved in our previous report.

To assess the mechanism of denaturation of fish muscle proteins during frozen storage, kinetic analysis of the rate of decrease in the solubility of the proteins was undertaken. Actomyosin solution isolated from carp muscle was frozen stored at various conditions of protein concentration, ionic strength, and pH, and the change of protein solubility was studied kinetically. Electron microscopic observations were also carried out to obtain information regarding changes in the shape of the actomyosin filaments. At pH 7.0, no single first order rate was found, but a two staged change consisting of two first order rate processes was illustrated regardless of the protein concentration and ionic strength. However, the mode of change varied depending on the pH. At a pH below 7.0, the change followed two staged process, while, at a pH above 7.5, it occurred in four successive stages, each of which was of a first order expression. From the results, three possible mechanisms have been proposed for the freeze denaturation of actomyosin.

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.

Textural differences in cooked fish muscle tissues, according to species, were demonstrated quantitatively by acquiring fresh tissue samples of five species, cooking them under specific conditions, measuring the common physical properties of each (drip, residual weight, fumness, cohesiveness, penetration, fiber volume, fiber diameter, and fiber length) and scoring them in a range of −1.2 to −1.8 for skipjack to 1.0 to 2.0 for channel rock fish using discriminant analysis.