Discrimination of Ca2+-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
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We have developed a procedure to discriminate actomyosin-type ATPase activity from Ca2+-ATPase activity of sarcoplasmic reticulum (SR) in mechanically skinned fibres, determining simultaneously their Ca2+-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 Ca2+-dependent ATPase activity, in spite of having little or no Ca2+-induced isometric tension. The residual ATPase activity is ascribed to the Ca2+-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 Ca2+-dependence of the ATPase activity is very similar to that of Ca2+-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 Ca2+-induced tension and actomyosin-type ATPase activity unchanged. This procedure indicated that the contribution of Ca2+-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 Ca2+-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 Ca2+-ATPase of SR. Ca2+-ATPase activity of SR in skinned fibres was inhibited completely by 10 μm CPA and held to one-half by about 0.2 μm. This effect was only partially reversible. CPA at 10μm or higher concentrations showed Ca2+-sensitizing action on myofibrils, which was readily reversible. CPA at 3μm inhibited almost completely the Ca2+-ATPase activity of SR, while it had no effect on either actomyosin-type ATPase or isometric tension of myofibrils.
KeywordsSkeletal Muscle Ionic Strength ATPase Activity Sarcoplasmic Reticulum Specific Inhibitor
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- Ebashi, S. &Endo, M. (1986) Ca ion and muscle contraction.Prog. Biophys. Mol. Biol. 18, 123–83.Google Scholar
- Goldman, Y. E. (1987) Kinetics of the actomyosin ATPase in muscle fibers.Ann. Rev. Physiol. 49, 637–54.Google Scholar
- Homsher, E. (1987) Muscle enthalpy production and its relationship to actomyosin ATPase.Ann. Rev. Physiol. 49, 673–90.Google Scholar
- Inesi, G. (1985) Mechanism of calcium transport.Ann. Rev. Physiol. 47, 573–601.Google Scholar
- Martonosi, A. N. &Beeler, T. J. (1983) Mechanism of Ca2+ transport by sarcoplasmic reticulum. InHandbook of Physiology, Section 10, Skeletal Muscle (edited by Peachey, L. D., Adrian, R. H. & Geiger, S. R.) pp. 417–85. Bethesda: American Physiological Society.Google Scholar
- Ogawa, Y. (1985) Sarcoplasmic reticulum: Ca-pump. InHandbook of Physiological Sciences, Vol. 4,Physiology of Skeletal, Cardiac and Smooth Muscles (edited by Tomita, T. & Sugi, H.) pp. 91–101. Tokyo: Igaku Shoin (in Japanese).Google Scholar
- Ogawa, Y. (1988) Comparative aspects of the mechanisms of energy transduction in sarcoplasmic reticulum between rabbit and frog skeletal muscle. InThe Roots of Modern Biochemistry (edited by Kleinkauf, H. Von Döhren, H. & Jaenicke, L.) Pp. 747–58. Berlin, New York: Walter de Gruyter.Google Scholar
- Ogawa, Y. &Kurebayashi, N. (1989) Modulations by drugs of the relationship between calcium binding to troponin C and tension. InMuscle Energetics (edited by Paul, R., Elzinga, G. & Yamada, K.) pp. 75–86. New York: Alan R. Liss.Google Scholar