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 -myosin heavy chains (-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% -MHC, whereas rat myocardium was induced to express 100% -MHC by surgical thyroidectomy and subsequent treatment with propylthiouracil. Slack test measurements at 15°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 -myosin than for pig -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 ϳ3.8-fold faster for rat -myosin (135 s ؊1 ) than for pig -myosin (35 s ؊1). ATP cleavage rates were nearly 30% faster for rat -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.There is considerable interest in the sequence and subunit composition of myosin isoforms due to the roles that variable expression may have in determining the contractile properties of myocardium and skeletal muscles. Type II myosin molecules are composed of two heavy chains (MHC) 1 (ϳ220 kDa each) and two pairs of light chains (MLCs) designated the essential (ϳ25 kDa) and regulatory light chains (ϳ16 kDa); both the MHCs and MLCs derive from multi-gene families (for review, see Ref.1). The expression of different myosin subunit isoforms confers a wide range of cross-bridge interaction kinetics in muscles of different types and from different species, variations that are ultimately due to divergence in the primary sequences of the encoding genes.One feature common to all myosins is the presence of two surface loops (loops 1 and 2) in the MHC subunit, but because of their hyper-flexibility, these loops were not resolved in the x-ray structure of chicken pectoralis myosin (2). Loop 1 is located in the vicinity of the catalytic site, and loop 2 is close to the actin-binding site. The sequences of the lo...
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