Katsuhiro Yamamoto

The binding of skeletal muscle C-protein to regulated actin

The binding of C‐protein, a component of thick filament of myofibrils, to regulated actin filaments in the presence or absence of CA2+ was studied. The amount of C‐protein bound to regulated actin filaments in the presence of CA2+ was higher than those in the absence of Ca2+. The addition of C‐protein to regulated actin caused an increase in turbidity, especially in the presence of Ca2+, and this was found to result from side‐by‐side association of actin filaments into bundles. In the absence of Ca2+, no actin filament bundles were formed.

Hydrostatic pressure-induced solubilization and gelation of chicken myofibrils

Hydrostatic pressure effect on chicken myofibrils was studied. Some of myofibrillar proteins were solubilized by pressure application above 200 MPa. Most of them were derived from thin filament; namely, actin, tropomyosin, and troponin components. Myosin heavy chain was hardly solubilized in 0.1 M NaCl, while its solubilization occurred in 0.2 M NaCl at 200–300 MPa. Solubilization of myosin dependend on magnitude of applied pressure and duration of treatment, and also salt concentration and pH. The maximal solubilization of myosin occurred at 200 MPa in 0.2 M NaCl at pH 7. Myofibrils in 0.1–0.2 M NaCl with a protein concentration of 40 mg/ml formed a gel by pressure application. The gel strength at 0.1 M NaCl increased with pressure, while it remained almost at the same level in 0.2 M NaCl. The intrinsic structure of myofibril was retained after pressure treatment in 0.1 M NaCl; however, pressure-induced disruption of myofibrillar structure occurred in 0.2 M NaCl.

Structural changes in chicken myosin subfragment-1 induced by high hydrostatic pressure

ATP-induced fluorescence increment of pressurized S1 was almost the same as unpressurized one at least up to 150 MPa, whereas it decreased above 200 MPa. The binding of e-ADP to S1 decreased at 250–300 MPa. S1 pressurized at 100–250 MPa and unpressurized S1 bound to F-actin similarly, although binding of S1 to actin decreased when the pressure treatment was done above 250 MPa. S1 was easily cleaved by tryptic digestion into three domains. Tryptic fragments of S1 digested after pressure treatment were essentially the same as those of unpressurized one. On the other hand, additional fragments appeared when the digestion was performed under pressure at 300 MPa. It is concluded that pressure-induced structural changes of S1 begin to occur about 150 MPa, and ATPase and actin binding sites lose those intrinsic structures at 250–300 MPa.

The role of sarcoplasmic protein in hydrostatic pressure-induced myofibrillar protein denaturation.

To observe the role of sarcoplasmic protein (SP) on myofibrillar protein (MP) denaturation under a hydrostatic pressure (HP), MP isolated from bovine muscle was treated with 300 MPa by increasing concentrations of SP (0, 0.8, 1.6, and 3.2 mg/ml) from bovine. SDS-PAGE patterns of soluble proteins in 0.1M NaCl (pH 7.4) indicated that a protein (about 100 kDa) from MP decreased with increasing concentrations of SP and that a 97 kDa protein from SP observed with 0.1 MPa was not observed with 300 MPa. SDS-PAGE patterns of soluble proteins in 0.6 M NaCl (pH 7.4) and Ca-ATPase activity showed that the denaturation of myosin heavy chain (MHC) was accelerated with increasing SP concentrations with the 300 MPa treatment. Thus, the addition of SP enhanced HP-induced denaturation of MHC and of a protein from MP of about 100 kDa.

A novel method for monitoring troponin T fragment from rabbit skeletal muscle during aging using quartz crystal microbalance.

BACKGROUND Troponin T (TnT) is degraded during aging of meat. The proteolytic fragment of TnT, especially the 30 kDa fragment, is used as one of indices for estimating aging of meat. We have tried to use quartz crystal microbalance (QCM), which is widely used to analyze interaction among macromolecules, to detect proteolytic fragments of TnT during aging of meat. RESULT The frequency of the QCM sensor with immobilized anti-TnT antibody in high-salt solution extracts of both myofibrils and whole meat decreased with time of aging. The staining intensity of the bands, including a 30 kDa fragment bound to anti-TnT antibody, also increased with time of aging in western blotting. These results confirm that TnT is degraded during aging and released from thin filaments, and QCM analysis is sufficiently sensitive to detect the TnT fragments. CONCLUSION The QCM analysis of muscle and myofibrillar extracts using anti-TnT antibody-immobilized sensor can be used as a convenient tool for monitoring the extent of aging of meat.

Changes in myosin molecule and its proteolytic subfragments induced by high hydrostatic pressure

Astract Turbidities of myosin, heavy meromyosin (HMM), and S1 increased with pressure. Whereas, light meromyosin (LMM) and rod did not show turbidimetric change. Head to head association forming a cluster was observed in HMM as well as myosin. S1 aggregate was larger than that of myosin or HMM. There was no noticeable morphological change in pressurized LMM and rod. S1 was split from unpressurized myosin by chymotryptic digestion in the absence of Ca 2+ . After pressurization of myosin, S1 splitting was suppressed. Hydrophobicities of myosin, HMM, and S1 increased with pressure, while no hydrophobic changes were detected in LMM and rod.

Immunochemical analysis of porcine cardiac C-protein.

C-protein has been isolated from pig heart and its immunochemical properties studied. It is extracted with myosin, and separated from the myosin on a DEAE-Sephadex column. The amount of C-protein recovered from crude myosin is approx. 3.5%. The molecular weight of C-protein is 150,000. Anti-C-protein serum reacts with crude myosin and purified C-protein but not with purified myosin in immunodiffusion plates. Cardiac C-protein does not react with anti-skeletal white muscle C-protein serum. Immunoblotting experiments show that anti-cardiac C-protein serum reacts with a Mr = 150,000 component in myofibrils or crude myosin. C-protein is located in the A-band, except the M-line region, of the myofibrils. These results indicate that C-protein is an intrinsic component of the thick filaments in pig heart myofibrils.