The complex of tropomyosin and troponin binds to actin and inhibits activation of myosin ATPase activity and force production of striated muscles at low free Ca 2+ concentrations. Ca 2+ stimulates ATP activity, and at subsaturating actin concentrations, the binding of NEM-modified S1 to actintropomyosin-troponin increases the rate of ATP hydrolysis even further. We show here that the Δ14 mutation of troponin T, associated with familial hypertrophic cardiomyopathy, results in an increase in ATPase rate like that seen with wild-type troponin in the presence of NEM-S1. The enhanced ATPase activity was not due to a decreased incorporation of mutant troponin T with troponin I and troponin C to form an active troponin complex. The activating effect was more prominent with a hybrid troponin (skeletal TnI, TnC, and cardiac TnT) than with all cardiac troponin. Thus it appears that changes in the troponin-troponin contacts that result from mutations or from forming hybrids stabilize a more active state of regulated actin. An analysis of the effect of the Δ14 mutation on the equilibrium binding of S1-ADP to actin was consistent with stabilization of an active state of actin. This change in activation may be important in the development of cardiac disease.Muscle contraction is a cyclic interaction of myosin and actin driven by the hydrolysis of ATP. Regulation of ATP hydrolysis in mammalian cardiac and skeletal muscle is mediated by four actin-associated proteins. Tropomyosin binds to seven actin monomers, and each tropomyosin is bound to a troponin complex consisting of troponin I, troponin T, and troponin C (TnI, 1 TnT, and TnC, respectively). The ATPase rate of myosin, in the presence of regulated actin, is cooperatively activated by Ca 2+ and by ATP-free forms of myosin. The rate of ATPase activity in the absence of Ca 2+ is low. Ca 2+ increases the k cat by ~18-fold and decreases the concentration of actin required for 50% activity by about 2-fold (1, 2). The ATPase rate can be increased further (≈8-fold) by the binding of "activating" myosin (myosin-ADP, nucleotide- † Supported by NIH Grant AR40540 (to J.M.C.), a grant from the American Heart Association (to S.F.), and NIH Grant HL63974 (to P.B.C.). A preliminary report of these data was presented at the 45th Annual Biophysical Meeting, Baltimore, MD, February 2004. * Corresponding author. Tel: 252-744-2973. E-mail:chalovichj@mail.ecu.edu.. ‡ East Carolina University. § Medical School Hannover. || Florida State University.1 Abbreviations: EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; NEM, N-ethylmaleimide; regulated actin, actin-tropomyosin-troponin; S1, myosin subfragment 1; SD, standard deviation; SEM, standard error of the mean; TnT, troponin T; TnI, troponin I; TnC, troponin C. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2006 January 25. Published in final edited form as:Biochemistry. 2004 December 7; 43(...

There is no consensus on the mechanism of inhibition of actin-myosin ATPase activity by caldesmon. Various models are based on different assumptions for the number of actin monomers that constitute a caldesmon binding site. Differences in binding behavior may be due to variations in the assay, the range of caldesmon concentrations, the type of caldesmon, and the method of data analysis used. We have evaluated these factors by measuring binding in the presence and absence of tropomyosin with both intact caldesmon and a recombinant 35 kDa actin binding fragment and with actin initially in the polymerized state or monomeric state. In all cases caldesmon binding could be simulated with a model having one class of binding sites. However, the number of actin monomers constituting a site was variable. Binding to F-actin at 165 mM ionic strength was best described with 7 actin monomers per site. When caldesmon bound to actin during the polymerization of G-actin, the size of the binding site was 3. Binding of the expressed truncated fragment, Cad35, could be described with 3 monomers per site. A simple interpretation of the data is that caldesmon binds tightly to 2-3 actin monomers. Additional parts of caldesmon bind less tightly to actin, causing caldesmon to cover approximately 7 actin monomers. The appendix contains an analysis of several binding curves with multiple binding site models. There is no compelling evidence for two classes of binding sites.

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