Atsushi Ooi

Insight into nucleotide and Ca2+ binding to carp α‐actin

Nucleotides and Ca2+ binding to α-actin prepared from ordinary skeletal muscle of carp Cyprinus carpio was studied. When bound Ca2+ was removed with ethylenediaminetetraacetic acid, carp α-actin denatured more rapidly than chicken α-actin. Kinetic studies of the denaturation process showed that in the absence of divalent cations, the binding constants of ATP to carp and chicken actin were 5.0×104/M and 1.2×105/M, respectively. Competitive binding of Ca2+ between actin and 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraacetic acid (Quin 2) showed that affinity of Ca2+ for carp actin was also lower than that for chicken actin by a factor of 1.6. These results indicated that carp actin could relatively easily denature due to the low affinities of these ligands. Enthalpy changes upon ATP binding to carp and chicken actin were −65 kJ/mol and −110 kJ/mol, respectively. Thermodynamic analyses of our results revealed that the entropy change associated with ATP binding to carp actin was significantly smaller than that to chicken actin, suggesting that structural stabilization upon ATP binding was less effective in carp actin.

Thermal stability of carp G-actin monitored by loss of polymerization activity using an extrinsic fluorescent probe

The thermal stability of carp G-actin was investigated by monitoring loss of actin polymerization ability. To determine the amount of native actin remaining after heat treatment, actin was labeled with a fluorescence reagent, N-(1-pyrene)iodoacetamide. The loss of polymerization ability of carp actin during heat treatment, at between 45 and 55°C, occurred faster than that of chicken actin. The inactivation rate was influenced by concentrations of ATP and Ca2+ in solution. With the increase of Ca2+ concentration, the inactivation of carp actin was markedly suppressed. Furthermore, the activation energy of the inactivation of carp actin obtained from an Arrhenius plot was similar to that of chicken actin. These results indicated that the thermal instability of carp G-actin was due to the low affinites of ATP and Ca2+ for carp actin described in a previous report.

Mechanochemical properties of ordinary and dark muscle myosins from seawater fish

Myosin was isolated from two types of muscle, ordinary and dark muscles, of three species of fish living in sea water. The compositions of light chains were visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the mechanochemical activity was examined by in vitro motility and ATPase assays. Ordinary muscle myosin of either species had three species of light chain, whereas dark muscle myosin had another two species of light chain judged by SDS-PAGE. Sliding velocity of ordinary muscle myosin was in the range of 4.92–6.89 μm/S, whereas that of dark muscle myosin was in the range of 3.07–4.25 μm/s. Therefore, ordinary muscle myosin showed 1.26–1.95 times higher sliding velocity than dark muscle myosin in either species. The ratios of Vmax of actin-activated Mg2+-ATPase activity of ordinary to dark muscle myosins were correlated quite well to the ratios of sliding velocity. Activity of ordinary muscle myosin was comparable to that of mammalian fast muscle myosin, but that of dark muscle myosin was twice of that of mammalian slow muscle myosin. These results may reflect the essential role of fish dark muscle myosin always used in slow cruising.

Seasonal Changes in Rigor Development and Flesh Texture of Wild Japanese Sea Bass (Lateolabrax japonicus)

ABSTRACT The season for finding the firmest sashimi made from wild Japanese sea bass was investigated. Flesh rigor tension and contraction were measured by a “new” device with isometric (muscle length kept constant) and isotonic (balance beam moves freely) transducers. Both flesh rigor tension and contraction peaks appeared at the same postmortem times, with attainment of ultimate pH and adenosine triphosphate/inosine monophosphate ratio in four seasons. Seasonal seawater temperature correlated negatively to seasonal maximum rigor contraction and positively to seasonal flesh breaking strength at 72 h postmortem. After rigor peak tension attainment, the tension relaxation was much lower than that of other wild fish species, especially in summer. The sea bass muscle resisted structure disruption in rigor isometric tension generation. Wild sea bass sashimi was firmest in breaking strength in summer.

C-protein (MyBP-C) isoforms from carp ordinary and dark muscles and muscle type-specific binding to myosin

C-protein is a myosin-associated protein of vertebrate striated muscle, and its function and properties have been extensively examined. However, there has been no report of C-protein of fish skeletal muscle so far. C-protein was identified in carp skeletal muscle by immunoassay using antibody against chicken C-protein, and the muscle-type specific C-protein was purified from carp ordinary and dark muscles for the first time. Although C-protein could be prepared from crude myosin by the reported procedure, C-protein degraded appreciably during the purification steps. Accordingly, C-protein was selectively extracted from the muscle with 0.15 M K-phosphate buffer (pH 5.8), and purified by ammonium sulfate fractionation, followed by AF-blue chromatography. Myosin free from the accessory proteins was obtained by diethylaminoethyl (DEAE) chromatography and used to assay the binding of C-protein with myosin. Ordinary muscle C-protein bound to ordinary muscle myosin in a saturable manner, but its maximum amount of binding was approximately twice that of dark muscle myosin. Similarly, dark muscle C-protein bound to dark muscle myosin much more than to ordinary muscle myosin. These results suggest that C-protein isoforms specifically bound with myosin isoforms originated from the same type of muscle.

Thermal stability of carp actin in its polymerized form

We investigated the thermal denaturation of carp F-actin by measuring the loss of polymerization ability. The thermal denaturation rates of carp F-actin were at least 10-fold higher than those of chicken F-actin. Binding of tropomyosin thermally stabilized carp F-actin with no appreciable change in activation energy, suggesting the instability was caused by a high frequency of fragmentation of the actin filaments. A comparison of the critical concentration for polymerization suggested that the subunit–subunit interactions of carp F-actin were indeed weaker than those of chicken F-actin. Furthermore, using fluorescence quenching we showed that the nucleotide binding region of carp F-actin was in a more open conformation. Together, our results suggest that the instability of carp F-actin is a function of both the rate of fragmentation and the dissociation of bound nucleotides.