Responsibility of myosin S-1 and rod for the polymerization of myosin heavy chain through disulfide bonding upon heating of actomyosin

To elucidate the role of S-1 and rod in the polymerization of myosin heavy chain through SS bonding upon heating of actomyosin, each myofibril of carp (with 100 mu g/ml leupeptin), flying fish and rabbit was digested by propo rtional to-chymotrypsin to S-1 and rod followed by heating for 1 h at tempe ratures ranging from 10 to 80 degrees C with added Cu2+ 12 mu M for carp an d 100 mu M for other samples to enhance the oxidation of SH groups. SDS-PAG E and the measurement of SH content and surface hydrophobicity were perform ed. Behaviors of S-1 and rod from 3 species in common were as follows. With the rising of temperature above 30 degrees C, polymer at the top of 3% gel through SS bonding gradually increased with the concomitant decrease i n S-l and actin with Cu2+, while less polymer was found without Cu2+. This polymer was confirmed to be composed of S-1 and less amount of actin by two -step SDS-PAGE. Rod dimer through SS bonding was found almost at any heatin g temperatures except at 50 degrees C where the degradation of rod occurred strongly. Surface hydrophobicity increased clearly from 30 degrees C, followed by gra dual increase above 50 degrees C. This behavior coincided very well with th e increasing behavior of RSH upon heating without Cu2+. Oxidation of TSH wa s promoted with added Cu2+ Slightly up to 30 degrees C and greatly above 30 degrees C, while RSH did so only a little below 30 degrees C, and then gre atly above 40 degrees C in flying fish and 50 degrees C in rabbit. It was, therefore, concluded that the polymer formation of HC upon heating of actomyosin was mainly due to the oxidation of SH groups in S-1, while th e dimer formation of HC was due to the oxidation of SH groups in rod.