Kinetic differences in cardiac myosins with identical loop 1 sequences

The kinetics of nucleotide turnover vary considerably among isoforms of ver tebrate type II myosin, possibly due to differences in the rate of ADP rele ase from the nucleotide binding pocket. Current ideas about likely mechanis ms by which ADP release is regulated have focused on the hyperflexible surf ace loops of myosin, i.e, loop 1 (ATPase loop) and loop 2 (actin binding lo op). In the present study, we investigated the kinetic properties of rat an d pig beta -myosin heavy chains (beta -MHC) in which we have found the sequ ences of loop 1 (residues 204-216) to be virtually identical, i.e, DQSKKDSQ TPKG, with a single conservative substitution (rat E210D pig), Pig myocardi um normally expresses 100% beta -MHC, whereas rat myocardium was induced to express 100% beta -MHC by surgical thyroidectomy and subsequent treatment with propylthiouracil. Slack test measurements at 15 degreesC yielded unloa ded shortening velocities of 1.1 +/- 0.8 muscle lengths/s in rat skinned ve ntricular myocytes and 0.35 +/- 0.05 muscle lengths/s in pig skinned myocyt es. Similarly, solution measurements at the same temperature showed that ac tin-activated ATPase activity was 2.9-fold greater for rat beta -myosin tha n for pig beta -myosin, Stopped-flow methods were then used to assess the r ates of acto-myosin dissociation by MgATP both in the presence and absence of MgADP. Although the rates of MgATP-induced dissociation of acto-heavy me romyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was similar to3.8-fold faster for rat beta -myosin (135 s(-1)) than for pig beta -myosin (35 s(-1)). ATP cleavage rates were nearl y 30% faster for rat beta myosin, Thus, whereas loop 1 appears from other s tudies to be involved in nucleotide turnover in the pocket, our results sho w that loop 1 does not account for large differences in turnover kinetics i n these two myosin isoforms, Instead, the differences appear to be due to s equence differences in other parts of the MHC backbone.