Physicochemical and Enzymatic Studies on Cardiac Myosin A

Cardiac myosin was isolated in ultracentrifugally homogeneous form from dog hearts and subjected to physicochemical and enzymatic studies. Distinct differences in molecular and enzymic properties were observed between cardiac and skeletal myosins: the molecular weight of cardiac myosin was found to be 758,000, by Archibald ultracentrifugation, as opposed to 500,000 to 619,000 for skeletal myosin; the adenosinetriphosphatase (ATPase) activity for the cardiac enzyme (V_max, 4 μmoles PO4/g enzyme/sec) was about one-third the value commonly observed for its skeletal counterpart.

The secondary structural characteristics of cardiac and skeletal myosin were essentially the same: the helical content of cardiac myosin, as inferred from optical rotatory dispersion, is 58%. However, some structural differences exist between the two molecules, as evidenced by their different behavior toward attack by trypsin. The cardiac protein did not yield products analogous to the meromyosins, and the digestion process was characterized by a slow progressive degradation of the native molecule to relatively lower molecular weight peptides.

Adenosinetriphosphatase activity and hydrodynamic and optical rotatory properties of cardiac myosin A were studied in aqueous solutions of ethylene glycol. This solvent caused a significant increase in ATPase activity at relatively high solvent concentrations (45 vol%), followed by a sharp decrease at higher concentration. Optical rotatory dispersion studies indicated pronounced changes in the conformation of myosin exposed to ethylene glycol in an amount corresponding to maximal ATPase activity. The disruption of hydrophobic bonds seems to be the cause of the collapse of the native α-helix structure, as well as being intimately involved in ATPase activation

Hydrodynamic, optical rotatory, and enzymic studies carried out on solutions of cardiac myosin A and myosin B containing ouabain suggested that the inotropic effect exerted by this glycoside is not mediated through direct alteration of the contractile protein (e.g., in the form of a size or shape change). Nor is any alteration produced in the ATPase active site of myosin to result in either an increased or a decreased rate of ATP hydrolysis.

In the final section of the paper, an attempt is made to integrate some of the hydrodynamic findings reported for cardiac myosin with the electron microscopic data of Huxley⁷ and of Spiro,⁸ in order to present a proposed model for myosin in the cardiac myofilament. From such an analysis, it is proposed that the cardiac myofilament contains 56 myosin molecules, a smaller number than the 370 molecules/filament previously calculated by Holtzer and associates¹³ for skeletal myosin. This difference is a direct reflection of the differences in molecular weight observed for the two molecules and the different dimensions of skeletal and cardiac fibrils used in the calculations.