Toshiyuki Fukazawa

Direct interactions between talin and actin

Talin was purified from chicken gizzard by a modification of the method of L. Molony et al. [J. Biol. Chem.(1987) 262, 7790-7795]. Unlike the talin purified by the previous method, the talin purified by the new method was found to bind to both F- and G-actin: Talin cosedimented with F-actin. On gel filtration of a mixture of talin and G-actin, a complex of talin and action was obtained. Talin stimulated the polymerization rate of G-actin. A major proteolytic fragment of talin that retained the binding ability to F-actin was also identified. These results indicate that talin can bind directly to actin and suggest that talin plays a key role in the organization of actin filaments at the actin-membrane attachment sites in vivo also.

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.

Effect of ATP concentration and pH on rigor tension development and dissociation of rigor complex in glycerinated rabbit psoas muscle fiber

Isometric rigor tension development of glycerinated rabbit psoas muscle fibers in a medium, due to the formation of rigor complexes, was estimated at varying ATP concentrations from 0 to 2.5 mM and pH values from 6.75 to 8.20. The dissociation of rigor complexes was also estimated under the same conditions. When muscle fibers developed rigor tension from the relaxed and rigor states, the magnitude of rigor tension increased with increasing concentration of ATP. Transition between rigor and relaxation in single fibers occurred discontinuously at constant levels (critical levels) of ATP which were determined by pH. The critical concentrations of ATP necessary for inducing the transitions between rigor and relaxed states were also increased exponentially with increased pH. Incomplete repetition of tension development by the same fiber was also observed. This incomplete reversibility was divided into two types: one which showed a decay in rigor tension and another which showed no decay. The reason for the incomplete reversibility was discussed

Observation of Transmitted Light Spectra during Gelation Process of Actomyosin

The heat-induced gelation process of muscle protein, actomyosin, was investigated by observing the transmitted light spectra. First, the temperature characteristic of the gelation ( T g ′ ) was determined from the spectral changes, showing remarkable dependence on pH and [NaCl]. Moreover, the spectra varied drastically with time after the temperature-jump procedure. From the time dependence of the observed spectra, the turbidity was calculated to characterize the light-scattering properties. The logarithm of the turbidity showed approximately linear dependence on the logarithm of the wavelength, and the slope increased with time, suggesting the growth of the actomyosin assembly during the gelation process.

Gelation process of actomyosin

The heat‐, and pressure‐induced gelation process of actomyosin was investigated by observing the transmitted light spectra. First, pH and [NaCl] dependences of the characteristic temperature (T’g) related to the gelation were determined at atmospheric pressure. Then, the temperature variations of the transmitted light spectra were monotored after the temperature‐ and pressure‐jump procedures, revealing the growth of the actomyosin assembly during these gelation processes.The heat‐, and pressure‐induced gelation process of actomyosin was investigated by observing the transmitted light spectra. First, pH and [NaCl] dependences of the characteristic temperature (T’g) related to the gelation were determined at atmospheric pressure. Then, the temperature variations of the transmitted light spectra were monotored after the temperature‐ and pressure‐jump procedures, revealing the growth of the actomyosin assembly during these gelation processes.

Preparative study on the isolation of single sarcomeres from rabbit skeletal muscle.

Single sarcomeres were prepared from fresh rabbit myofibrils by digestion with a calcium-activated factor (CAF). The rabbit single sarcomere has functional properties quite similar to those of single sarcomeres obtained from chicken muscle by a usual method. Thus it was found that the single sarcomeres obtained by CAF digestion were useful as a muscle model, though they were not completely intact.