Preparation of heavy meromyosin from the autolyzed squid mantle muscle homogenate

ABSTRACT:   Incubation of squid mantle muscle homogenate caused a selective cleavage of myosin into heavy meromyosin (HMM) and light meromyosin (LMM). HMM was isolated from the incubated homogenate by using ammonium sulfate fractionation. The purified HMM retained two types of light chain components. Its Mg²⁺-ATPase activity with or without F-actin showed a Ca-sensitivity. HMM was cleaved into subfragment-1 and subfragment-2 upon chymotryptic digestion with or without Ca²⁺, possessing different light chain composition. Two types of light chain component were kept intact when digested in the presence of Ca²⁺. Ca²⁺ stabilized HMM especially in a bound form to F-actin.     

Heat-induced structural changes and aggregation of walleye pollack myosin in the light meromyosin region

Heat-induced structural changes and aggregation properties of walleye pollack myosin, light meromyosin (LMM) and heavy meromyosin (HMM) were investigated. According to the circular dichroism (CD) measurement, the α-helix content of the pollack myosin and LMM were estimated to be 72% and 90% at 5°C but decreased to 22% and 21% by increasing the temperature to 60°C with two transitions at 35°C and 50°C, respectively. In contrast, that of HMM decreased gradually from 37% to 33% by increasing the temperature from 5°C to 40°C, and decreased steeply to 20% above 50°C. These results indicate that the decrease in the α-helix content in the myosin molecule upon heating was attributable mainly to the decrease in the α-helix content in the LMM region. In contrast, 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence and light scattering intensity of both myosin and HMM were remarkably increased above 25°C and 35°C, respectively, while those of LMM showed only a slight change even above 60°C. Although LMM alone formed no aggregates detectable by the light scattering measurement, it formed coprecipitates with myosin but not with HMM upon heating at 40°C for 10 min. These facts suggest that LMM bind to the LMM region of the myosin. Further, it was found that myosin gel formed in a test tube by the same heating conditions was significantly weakened by coexistence of LMM. These results suggest that the association of the LMM region of myosin molecules is essential for the heat-induced gelation of myosin.     

cDNA cloning and complete primary structures of myosin heavy chains from brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso fast skeletal muscles

ABSTRACT:   The complete cDNA sequences encoding predominant types of myosin heavy chain (MYH) in the fast skeletal muscle were determined for brushtooth lizardfish Saurida undosquamis and wanieso lizardfish S. wanieso , which are used as materials for preparing high-quality surimi-based products. The cDNA consisted of 5973 and 5987 bp, respectively, and both encompassed an open reading frame encoding a polypeptide of 1936 amino acid residues. Brushtooth and wanieso lizardfish MYH showed the amino acid sequence identity of 92–93% to white croaker MYH, which was higher than that of 90% to walleye pollack MYH. The putative binding sites for ATP, actin, and regulatory and essential light chains in the subfragment-1 region of brushtooth lizardfish MYH exhibited a high identity with white croaker counterparts as well as the sequences of subfragment-2 and light meromyosin. In contrast, phylogenetic tree, constructed by the neighbor-joining method based on mitochondrial 16S rRNA gene, revealed that the two lizardfish species formed a cluster with walleye pollack, which was paraphyletic with white croaker. Therefore, a good reputation for lizardfish and white croaker to have a high thermal-gel forming ability seemed to be reflected by MYH rather than biological similarity as revealed by the mitochondrial 16S rRNA gene.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.     

Myofibrillar proteolysis by myofibril-bound serine protease from white croaker <Emphasis Type="Italic">Argyrosomus argentatus</Emphasis>

ABSTRACT:   The modori phenomenon is defined as heat-induced myofibrillar degradation caused by endogenous serine protease(s) of fish muscle during Kamaboko fish meat gel production. This study was undertaken to analyze myofibrillar proteolysis of white croaker Argyrosomus argentatus muscle, which is an ingredient of high quality Kamaboko, by myofibril-bound serine protease (MBSP) under conditions corresponding to the modori phenomenon. White croaker MBSP was stable between pH 2–11 and below 65°C, and about 60% of its initial activity remained after incubation for 2 h under the conditions at 65°C and pH 7.5. About 60% of the enzyme activity was suppressed by 0.5 M NaCl. White croaker MBSP degraded various myofibrillar proteins between 40 and 70°C and pH 6.0–9.0, and preferentially degraded myosin heavy chain rather than other myofibrillar proteins. The enzyme degraded the myosin heavy chain most strongly at 55°C and pH 7.0, and a major part of the bands of myosin heavy chain and its degradation products disappeared for a period of 2 h. These degradation characteristics are very similar to those observed during the modori phenomenon, indicating that MBSP could be a modori-inducing protease involved in the modori phenomenon of white croaker Kamaboko production.     

Thermal denaturation and autolysis profiles of myofibrillar proteins of mantle muscle of jumbo squid Docidicus gigas

ABSTRACT:    Jumbo squid was very similar to Japanese common squid in terms of myofibrillar Ca²⁺-, Mg²⁺- and K⁺(EDTA)-ATPase activities. Myofibrils of jumbo squid were significantly stabilized upon addition of Ca²⁺ and destabilized by increasing KCl concentration for heating. Incubation of muscle homogenate of jumbo squid induced a selective cleavage of myosin into two major fragments and the cleavage was inhibited by EDTA. Autolysis was prominent at and above 0.3 M NaCl where myosin filaments dissolve. The enzyme involved in the autolysis was proved to be unstable showing maximal autolysis rate at 25°C. Washing the homogenate partially reduced the autolysis activity.     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.     

Stabilization of myosin by ionic compounds as affected by F-actin

ABSTRACT:   Suppressive effects of non-ionic (sorbitol, maltose, and trehalose) and ionic (Na-glutamate, Na-acetate, Na-sulfate, and ammonium sulfate) compounds on the thermal inactivation of myosin subframgent-1 (S-1) and myofibril Ca²⁺-ATPase were compared. All compounds suppressed S-1 denaturation. When myofibrils were used (at 0.1 M KCl), sugars and sugar alcohol (non-ionic compounds) suppressed denaturation similar to S-1, while Na-glutamate, Na-acetate, and Na-sulfate weakly suppressed them. Ammonium sulfate accelerated denaturation, but suppressed denaturation when heated in 2 M KCl, at which myosin lost protection by F-actin. It was thus concluded that ionic compounds affected the denaturation of myofibrils in two ways; suppression as established with S-1, and acceleration as a result of loss of protection by F-actin caused by increase in ionic strength.     

Effect of pH on the gelation of walleye pollack surimi and carp actomyosin pastes

ABSTRACT: The effect of pH on thermal gelation and transglutaminase (TGase; EC2.3.2.13)-induced suwari (setting) of surimi and actomyosin pastes was investigated. A strong and elastic gel was produced from walleye pollack surimi paste at pH 7.0 in the presence of Ca²⁺ using a two-step heating method. In contrast, walleye pollack actomyosin paste formed a weak gel under the same conditions as a result of the low concentration of endogenous TGase. In the presence of EGTA [ethyleneglycol bis(2-aminoethylether) tetraacetic acid], weak gels were formed at pH values of 7.0 and 6.0. Non-proteolytic modori (gel weakening) occurred extensively in the course of actomyosin gelation, but not in surimi gelation. Maximum TGase-induced myosin heavy chain cross-linking was observed at a slightly higher pH of 7.5 than at the optimal pH of endogenous TGase activity; the difference being derived from different substrates. Gelation of carp actomyosin paste at pH values of 5.5, 6.0, 6.5 and 7.0 was monitored by measuring storage modulus (G') and loss modulus (G"). A weak gel was formed at all pH values, but a slightly rigid and less elastic gel was obtained at lower pH values. The addition of microbial TGase (MTGase) formed strong elastic gels at pH 7.0 and 6.5. MTGase cross-linked myosin heavy chains even at pH 5.5, but contributed neither to suwari response nor strong gel formation. Overall, results suggest that the optimal pH for the gelation of surimi paste from easy-setting fish species is a compromise between the pH-optima of TGase activity and of preferable actomyosin conformation for myosin cross-linking.     

Comparison of characteristics of actomyosin from white, pink, and red muscle fiber types in cultured carp

ABSTRACT: The present study compared and examined the characteristics of actomyosin among white (W), pink (P), and red (R) muscle fiber types in carp (cultured). Both the superprecipitation reaction and the Mg²⁺-ATPase activity of actomyosin became higher with increased Ca²⁺ concentration (pCa 7.0–pCa 5.0) and with decreased adenosine triphosphate (ATP) concentration (3.0–0.5 mM) in all three muscle fiber types. A comparison of the three fiber types shows that the superprecipitation reaction of actomysoin was lower in the order of W < P < R and, in contrast, was higher for Mg²⁺-ATPase activity in the order of W > P > R. A significantly positive correlation between both values was found for each of the three muscle fiber types, but these correlations were clearly different among the three muscle fiber types, and the superprecipitation reaction of actomyosin was lower in the order of W < P < R when Mg²⁺-ATPase activity was at the same level. In conclusion, the characteristics of actomyosin were remarkably different among white, pink, and red muscle fiber types.     

The existence of aspolin and its trimethylamine-<Emphasis Type="Italic">N</Emphasis>-oxide demethylating activity in the muscle of freshwater fish

ABSTRACT:   Aspolin is a polyaspartic acid-like protein, which is originally isolated from walleye pollack Theragra chalcogramma muscle as trimethylamine- N -oxide (TMAO) demethylase. Although carp Cyprinus carpio muscle contains a trace amount of the enzyme substrate, TMAO, aspolin can be extracted and purified by acid treatment, successive chromatographies and polyacrylamide gel electrophoresis, and has twice the amount of that in walleye pollack muscle. Carp aspolin showed a low enzymatic activity in the presence of Fe²⁺ and reductants, and its Km value (100 mM) to TMAO was extremely high. It was a thermostable protein and had an unfolded conformation. The amino acid sequence of carp aspolin 1 deduced from cDNA revealed that it contained a long Asp polymer, an uninterrupted stretch of 138 Asp residues, followed by four amino acid residues, His-Glu-Glu-Leu, in C-terminus. The chain length was shorter by 42 Asp residues than that of its walleye pollack counterpart.     

Characterization of 38 kDa and 42 kDa chitinase isozymes from the liver of Japanese common squid Todarodes pacificus

ABSTRACT: Characterization was investigated on the 38 kDa and 42 kDa chitinase (EC3.2.1.14) isozymes from the liver of Japanese common squid Todarodes pacificus . Optimum pH toward colloidal chitin was observed at pH 3.0 for the 38 kDa chitinase, and pH 3.0 and 9.0 for the 42 kDa chitinase. K _m and k _cat of the 38 kDa and 42 kDa chitinases toward a longer substrate, glycol chitin, were 0.071 mg/mL and 1.22/s, and 0.074 mg/mL and 0.196/s, respectively. Alternatively, strong substrate inhibition of both chitinases were observed toward a short substrate, N -acetylchitopentaose (GlcNAc₅). Both chitinases decomposed not only chitin but also chitosan (D. A. 95%). The cleavage pattern and reaction rate were investigated using N -acetylchitooligosaccharides (GlcNAc_n, n  = 2–6). Both chitinases hydrolyzed GlcNAc_n ( n  = 4,5, and 6). The release of GlcNAc was not observed. The speed of the reaction was observed to be in the following order: GlcNAc₄ > GlcNAc₅ > GlcNAc₆ for the 38 kDa chitinase, and GlcNAc₆ > GlcNAc₅ > GlcNAc₄ for the 42 kDa chitinase. Both the chitinases released p -nitrophenol from p -nitrophenyl GlcNAc_n ( n  = 2, 3, and 4). N-terminal amino acid sequences of the 38 kDa and 42 kDa chitinases were YLLSXYFTNWSQYRPGAGKYFPQNI and EYRKVXYYTNWSQYREVPAKFFPEN, respectively.     

Thermal gelation properties of white croaker, walleye pollack and deepsea bonefish surimi after suwari treatment at various temperatures

The effects of setting (suwari) at around 40 °C on the breaking strength and breaking strain rate of thermal gels treated at 85 °C for 20 min during the following processing step were examined in association with the polymerization and degradation of myosin heavy chains (MHCs) for surimi prepared from white croaker, walleye pollack and deepsea bonefish. In the case of white croaker and walleye pollack, maximum values of breaking strength and breaking strain rate were obtained after suwari at 30–40 °C for both 30 and 60 min, at which temperature MHCs were polymerized. In comparison, these textual properties of the thermal gels decreased in surimi prepared from deepsea bonefish after suwari at around 38 °C for 30 min and at around 32 °C for 60 min, with concomitant degradation products of MHC. The textual properties of deepsea bonefish after suwari at temperatures >45 °C tended to be almost the same as those after suwari at temperatures of <30 °C, where neither polymerization nor degradation of MHC was observed.     

Comparative study on thermal denaturation modes of myosin in walleye pollack and carp myofibrils as affected by salt concentration

ABSTRACT:   The effect of salt concentration on the thermal denaturation profile of myosin in walleye pollack and carp myofibrils was compared by studying the subfragment-1 (S-1) and rod denaturation rates upon heating. Species-specific denaturation mode observed at 0.1 M KCl was no longer detected when samples were heated above 0.5 M KCl, where S-1 and rod denaturation rates were identical to each other. As the heating of the chymotryptic digest of myofibril formed practically no rod aggregates, S-1 denaturation in a form of myosin was the rate limiting step for rod aggregate formation. As the aggregate formation by rod was remarkably suppressed by lowering the temperature, the free movement of myosin tail upon heating was suggested to play an important role in the rod aggregate formation in a high salt medium.     

Purification and characterization of transglutaminase from squid gill

ABSTRACT: The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca²⁺ concentration, it was not sufficiently activated even by 50 mM CaCl₂ in the absence of NaCl, but could be fully activated with 10 mM CaCl₂ in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca²⁺ at pH 7.5–9.0 and had a rate constant (K _D ) of 1.6 × 10^–5 s^–1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca²⁺ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.     

Effects of environmental Ca2+ levels on branchial chloride cell morphology in freshwater-adapted killifish Fundulus heteroclitus

ABSTRACT: To examine the involvement of chloride cells in the uptake of Ca²⁺ in freshwater (FW) killifish, chloride cell morphology was compared in fish acclimated to different defined FW environments with Ca²⁺ concentrations of either 0.1 mM, 0.5 mM, or 2.5 mM. Numerous chloride cells were detected in whole-mount preparations of the gill filaments, which were stained with an antiserum specific for Na⁺, K⁺-ATPase. Chloride cells, located mostly on the afferent–vascular edge of the filaments, were larger at lower Ca²⁺ concentrations. Electron microscopic observations showed that in the 0.1 mM and 0.5 mM Ca²⁺ experimental groups, the apical membrane of chloride cells were flat or slightly projecting and equipped with numerous microvilli. In the 2.5 mM Ca²⁺ group, some chloride cells formed an apical pit, whereas other cells were similar to those observed in the 0.1 mM and 0.5 mM Ca²⁺ groups. Plasma osmolality decreased with decreasing ambient Ca²⁺ concentration, suggesting that environmental Ca²⁺ affects the permeability of the body surfaces. Gill Na⁺, K⁺-ATPase activity in the 0.1 mM and 0.5 mM Ca²⁺ groups were significantly higher than that in the 2.5 mM Ca²⁺ group, implying the involvement of the Na⁺–Ca²⁺ exchanger in Ca²⁺ uptake in the gills. These findings suggest that chloride cells function as the site for Ca²⁺ uptake in killifish acclimated to low Ca²⁺ environments.     

Binding characteristics of the Zn-binding membrane protein from common carp

ABSTRACT:    This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of ⁶⁵Zn to the 43 kDa protein-liposomes, and the binding of the ⁶⁵Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that ⁶⁵Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of ⁶⁵Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (N_max), 76.7 pmole/µg protein (approx. 3 mole of Zn²⁺/mole); and equilibrium dissociation constant (Kd), 0.19 µM. Of the cations introduced (Ca²⁺, Cd²⁺,Co²⁺, Cr²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Pb²⁺), only Co²⁺ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a N_max of 1.1 pmole/µg laminin, and Kd of 4.79 µM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.     

Purification and characterization of the amylase from a small abalone <Emphasis Type="Italic">Haliotis sieboldii</Emphasis>

ABSTRACT:   Amylase, with MW of 59 kDa, was purified from small abalone Haliotis sieboldii by ammonium sulfate fractionation, CM Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The optimal pH and temperature of purified amylase were 6.0 and 37°C, respectively. The purified enzyme was stable at pH 6.0–8.0 and low temperatures. It was activated by Ba²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Mn²⁺, K⁺, Ag⁺, Na⁺ and Li⁺, but completely or partially inhibited by Al³⁺, Cu²⁺, Cd²⁺, Hg²⁺ and Zn²⁺. EDTA could completely inhibit, while iodoacetamide, N-ethylmaleimide and urea partially inhibit the purified amylase. According to the digestion mode of various polysaccharides, the purified enzyme was considered to be an α-amylase.