Role of the sarcoplasmic reticulum in glycogen metabolism. Binding of phosphorylase, phosphorylase kinase, and primer complexes to the sarcovesicles of rabbit skeletal muscle.
Sarcoplasmic vesicles and beta-glycogen particles 30-40 mmicro in diameter were isolated from perfused rabbit skeletal muscle by the differential precipitation-centrifugation method. This microsomal fraction was subjected to zonal centrifugation on buffered sucrose gradients, in a B XIV Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 mmicro, proteins, glycogen, and enzymatic activities (phosphorylase b kinase, phosphorylase b, and glycogen synthetase) were performed. Whereas the entire synthetase activity was found combined with the glycogen particles, 39% of phosphorylase and 53% of phosphorylase b kinase activities, present in the microsomal fraction, were recovered in the purified vesicular fraction (d = 1.175). This latter fraction consists of vesicles, derived from the sarcoplasmic reticulum, and of small particles 10-20 mmicro in diameter attached to the outer surface of the membranes. These particles disappear after alpha-amylase treatment. Incubation of the sarcovesicular fraction with (14)C-labeled glucose-1-phosphate confirms the localization of a polysaccharide synthesis at the level of the membranes. "Flash activation" of phosphorylase b, i.e. Ca "activation" of phosphorylase kinase followed by a conversion of phosphorylase b into a, was demonstrated in the purified sarcovesicular fraction. Moreover, the active enzymatic sites were detected on the membranes by electron microscopy. The presence of binding sites between the membranes of the sarcoplasmic vesicles and a glycogen-enzyme complex suggests that this association plays a role in the glycogenolysis during muscle contraction.
D000818:Animals; D001665:Binding Sites; D002501:Centrifugation, Zonal; D005964:Glucosyltransferases / Q000378:metabolism*; D006003:Glycogen / Q000032:analysis / Q000378:metabolism*; D006651:Histocytochemistry; D008854:Microscopy, Electron; D008861:Microsomes / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology; D009119:Muscle Contraction; D009132:Muscles / Q000201:enzymology; D009702:Nucleoside Diphosphate Sugars; D010764:Phosphorylase Kinase / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*
D009702:Nucleoside Diphosphate Sugars; D006003:Glycogen; D005964:Glucosyltransferases; D010764:Phosphorylase Kinase