On the localization of sarcotubular ATPase activity in mammalian skeletal muscle

Summary

ATPase activity of the sarcoplasmic reticulum has been demonstrated at the level of the light microscope. Although this membrane system is usually viewed as ultrastructural in its dimensions, it was possible to identify sarcotubular enzymic activity in frozen sections. In skeletal muscle fibers of the rat diaphragm, sarcotubular ATPase can be distinguishedin situ from ATPases associated with mitochondria and myofibrils. This is possible because chemical properties are more readily analyzed in frozen sections than in material prepared for electron microscopy. Pour different ATPases have thus been localized in skeletal muscle fibers by taking advantage of differences in the pH optima of these enzymes and in their response to various inhibitors and activators. The following cytochemical and morphological features have been demonstrated:

• 1. While both mitochondrial and sarcotubular ATPases are active at pH 7.2 in the presence of cysteine, only mitochondrial ATPase activity survives when cysteine is replaced with the mercurial compound, PHMB. Two sarcotubular ATPases, on the other hand, survive formalin fixation under conditions which inhibit mitochondrial ATPase. Myofibrillar ATPase is also demonstrated in the presence of cysteine, but the pH optimum is closer to 9.4. This enzyme is both sulfhydryl dependent and formalin sensitive.

• 2. Although the spatial distribution of mitochondria and of sarcoplasmic reticulum in mammalian skeletal muscle fibers is similar, ATPases associated with these organelles can be distinguished by taking advantage of their differential response to mercurial and to formalin. In transverse section, sarcotubular ATPase activity is associated with a distinct, more or less continuous network surrounding myofibrils. This pattern differs from that formed by mitochondria, which are disposed in a less continuous array of filaments and granules. In longitudinal section, activity occurs at the site of the triads of the sarcoplasmic reticulum. If sections are fixed with formalin prior to incubation, an additional site of activity appears in the region of the H band. The morphological distribution of these two sarcotubular ATPases is distinguishable from that of both mitochondrial and myofibrillar ATPases.

These results suggest the possibility that the two sites of sarcotubular activity reflect two different roles of ATPase in this membrane system. Activity at the triads might be involved indirectly in making available the calcium necessary for muscular contraction, that is, by binding calcium which can be released at the time of contraction. Activity at the H bands might be more directly involved in the rebinding of calcium leading to relaxation of the muscle.