Purification of Cardiac Myosin from Rat Heart Proteolytic Cleavage and Its Inhibition

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Abstract
Using phenylmethanesulfonyl fluoride (PMSF) in all the steps of the preparation procedure to inhibit protease activity, purified cardiac myosin was isolated from rat hearts. The purified myosin obtained was free from nucleic acid, actin, tropomyosin, C-protein, and proteolytic products of myosin. Without PMSF, the myosin preparation containing proteolytic products was obtained. The properties of the ATPase of rat cardiac myosin [EC 3.6.1.3] were studied. The Ca2+−, K+− and Mg2+−ATPase activities of the purified myosin at high ionic strength and pH 7.0 were 1.15, 0.2, and 0.05 μmol P1 mg−1 min−1 respectively. Those of the myosin prepared without PMSF were lower than the above values and fluctuated from preparation to preparation. The Ca2+− activity of rat cardiac myosin was not activated by PCMB, although the content of SH groups was 6.6-7 mol per 105 g myosin. This anomalous property and the content of SH groups were also found in the myosin preparation containing proteolytic products. Analysis of subunits in the purified myosin was carried out on 3.5% acrylamide gels with 0.1% SDS. Of the total protein present in myosin, 10.2% was in the light chains; LC1 6.2% and LC2, 4.0%. Urea gel electrophoresis of rat cardiac myosin showed a band corresponding to LC1 and two bands coresponding to LC2 Without the protease inhibitor, rat cardiac myosin underwent proteolytic cleavage during its preparation, producing two fragments with molecular weights of 117,000 and 160,000. It is thought that one of them, the 117,000 fragment, is a constituent of subfragment 1 produced from cardiac myosin by inherent protease activity in rat heart cells.