Discrimination of Ca(2+)-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities.

We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca(2+)-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca(2+)-induced tension and accompanying ATPase activity. When they were treated with an alkaline CyDTA-containing solution of low ionic strength which was reported to remove troponin C, the fibres showed a considerable amount of Ca(2+)-dependent ATPase activity, in spite of having little or no Ca(2+)-induced isometric tension. The residual ATPase activity is ascribed to the Ca(2+)-ATPase activity of SR, because it is completely abolished by 1% CHAPS treatment for 10 min. This conclusion is also supported by the finding that the Ca(2+)-dependence of the ATPase activity is very similar to that of Ca(2+)-ATPase of SR isolated from rabbit skeletal muscle, and that the estimated activity is consistent with the reported values of direct determinations. On the other hand, treatment with a detergent such as CHAPS or Triton X-100 removes SR activities (ATPase and Ca-uptake), leaving Ca(2+)-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca(2+)-ATPase activity of SR may be minimal in total steady-state ATPase activity of mechanically skinned mammalian skeletal muscle fibres. Successive CyDTA and CHAPS treatments eliminated both Ca(2+)-induced tension and ATPase activity, which were recovered by the addition of troponin C. Using these procedures, we also examined the effect of cyclopiazonic acid (CPA) which was reported to be a specific inhibitor of Ca(2+)-ATPase of SR. Ca(2+)-ATPase activity of SR in skinned fibres was inhibited completely by 10 microM CPA and held to one-half by about 0.2 microM. This effect was only partially reversible. CPA at 10 microM or higher concentrations showed Ca(2+)-sensitizing action on myofibrils, which was readily reversible. CPA at 3 microM inhibited almost completely the Ca(2+)-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.