Kinetic differences in cardiac myosins with identical loop 1 sequences

The kinetics of nucleotide turnover vary considerably among isoforms of vertebrate type II myosin, possibly due to differences in the rate of ADP release from the nucleotide binding pocket. Current ideas about likely mechanisms by which ADP release is regulated have focused on the hyperflexible surface loops of myosin, i.e. loop 1 (ATPase loop) and loop 2 (actin binding loop). In the present study, we investigated the kinetic properties of rat and pig beta-myosin heavy chains (beta-MHC) in which we have found the sequences of loop 1 (residues 204-216) to be virtually identical, i.e. DQSKKDSQTPKG, with a single conservative substitution (rat E210D pig). Pig myocardium 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 degrees C yielded unloaded shortening velocities of 1.1 +/- 0.8 muscle lengths/s in rat skinned ventricular myocytes and 0.35 +/- 0.05 muscle lengths/s in pig skinned myocytes. Similarly, solution measurements at the same temperature showed that actin-activated ATPase activity was 2.9-fold greater for rat beta-myosin than for pig beta-myosin. Stopped-flow methods were then used to assess the rates of acto-myosin dissociation by MgATP both in the presence and absence of MgADP. Although the rates of MgATP-induced dissociation of acto-heavy meromyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was approximately 3.8-fold faster for rat beta-myosin (135 s(-)(1)) than for pig beta-myosin (35 s(-)(1)). ATP cleavage rates were nearly 30% faster for rat beta-myosin. Thus, whereas loop 1 appears from other studies to be involved in nucleotide turnover in the pocket, our results show that loop 1 does not account for large differences in turnover kinetics in these two myosin isoforms. Instead, the differences appear to be due to sequence differences in other parts of the MHC backbone.