Fact and artifact in the histochemical procedure for myofibrillar ATPase

Abstract

A recent quantitative biochemical study has shown that skeletal muscle mitochondria have high ATPase activity under the same conditions that are employed in the histochemical procedure for myofibrillar (actomyosin) ATPase. However, mitochondrial staining is not observed when this histochemical procedure is applied to frozen sections of skeletal muscle. The present investigation was performed to resolve this discrepancy. Frozen sections of muscle were incubated in the presence of inorganic phosphate (in place of ATP) and then treated sequentially with calcium chloride, cobalt chloride, and ammonium sulfide to determine whether inorganic phosphate binds to specific sarcoplasmic constituents. Binding of phosphate did occur and, furthermore, it was localized to the myofibrils and not at all to the mitochondria. It is concluded that the hydrolysis of ATP in the histochemical reaction can result from ATPase activity of myofibrils, mitochondria, or both. Since the released inorganic phosphate binds to the myofibril, the localization of the reaction product does not necessarily reveal all sites of enzymatic activity. Thus the histochemical procedure does not reflect myofibrillar ATPase activity exclusively, and one therefore cannot use this technique to infer that individual muscle fibers are physiologically fast or slow according to the intensity of the ATPase staining of their myofibrils.