CYTOCHEMISTRY OF PHOSPHATASES OF THE SARCOPLASMIC R E T I C U L U M II. In Situ Localization of the MG-Dependent Enzyme

The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17°C, in media containing 125 m~ Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgC12, and 1 mM Pb(NO3)2. After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoatc, and lack of inhibition by ouabain. In the, preceding paper, the presence of a Mgdependent, cation-stimulated ATPasO in a microsomal fraction of rabbit psoas muscle was reported, and some of its characteristics were described (1). The association of the enzyme with a sarcotubular fraction suggested that it was located somewhere within the sarcotubular system--either the T system, the sarcoplasmic reticulum, or possibly 1 Abbreviations used: ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; ITP, inosine triphosphate; CTP, cytidine triphosphate; UTP, uridine triphosphate; ADP, adenosine diphosphate; IDP, inosine diphosphate; AMP, adenosine monophosphate; NEM, N-ethyl maleimide; PCMB, p-hydroxymercuribenzoate; EGTA, ethylene glycol-bis(~-aminoethylether)-N, Nt-tetraacetic acid. at the junction between the two. In this study, the biochemical findings were used to construct a histochemical method for localization of the Mg-dependent enzyme. The way in which chemical activity was affected by histochemical reagents and incubation conditions was investigated. The final product of the Mg-dependent enzyme reaction was demonstrated, first in relation to the isolated vesicles of the sarcotubular fraction, and then in situ. M A T E R I A L S AND M E T H O D S P R E P A R A T I O N S U S E D : A microsomal fraction, consisting predominantly of smooth-surfaced vesicles when viewed in sectioned material, was isolated from rabbit psoas muscle as previously described (1). Only freshly prepared microsomes were used in histochem489 on O cber 9, 2017 jcb.rress.org D ow nladed fom