ROLE OF THE SARCOPLASMIC R E T I C U L U M IN GLYCOGEN METABOLISM Binding of Phosphorylase, Phosphorylase Kinase, and Primer Complexes to the Sarcovesicles of Rabbit Skeletal Muscle

Sarcoplasmic vesicles and ~-glycogen particles 30-40 m# 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 X I V Anderson type rotor, for 15 hr at 45,000 rpm in order to separate the two cytoplasmic organelles. Zonal profiles of absorbance at 280 m/~, 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 m~ in diameter attached to the outer surface of the membranes. These particles disappear after a-amylase treatment. Incubat ion of the sarcovesicular fraction with ~*C-labeled glucose-l-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 glycogenenzyme complex suggests that this association plays a role in the glycogenolysis dunng muscle contraction. I N T R O D U C T I O N Many efforts were devoted to extracting, identifying, and purifying the different cell constituents, and now complementary works strive to reconstitute integrated functional systems similar to those existing in living cells by recombining isolated elements Glycogen is an example among many others: it has been extracted from different tissues and then washed or treated to eliminate the contaminating substances. This last step in the purification is significant because the 3 -5% proteins 206 THE JOURNAL OF CEL~ BIOLOGY • VOLUME 64, 1972 • pages ~06-~24 on O cber 0, 2017 jcb.rress.org D ow nladed fom which "contaminate" glycogen prepared with mild methods @Vanson and Drochmans, 1968 a) represent mainly enzymes involved in the synthesis and breakdown of the polysaccharide. The binding of phosphorylase to glycogen has been established very early and its physicochemical properties have been studied by Madsen and Cori (1958). In a quanti tat ive approach to the distribution of phosphorylase in fractions isolated from liver homogenates, Ta ta (1964) showed that 70-80% of the activity was present in the microsomal fraction and that the majority of the recoverable enzyme was associated with the glycogen particles More recently, Meyer et al (1970) and, in the same laboratory, Heilmeyer et al (I970) extended the concept of an enzyme-glycogen complex by proposing an integrated functional system which includes particulate glycogen, phosphorylase, and phosphorylase kinase. A further relationship between particles of glycogen and membrane-bound structures in liver cells has been investigated with very critical assays by Luck (1961). Impressed by the morphological observations of Porter and Bruni (1959), he studied the distribution of the glycogen synthetase in the microsomal components extracted from a liver homogenate. He confirmed the observations of Leloir and Goldemberg (1960), according to which the enzyme involved in the synthesis of glycogen was firmly bound to the polysaccharide, and concluded decisively that the membranesof the smooth endoplasmic ret lculum were not directIy engaged in the process In our laboratory, we met similar problems of distribution and functional association of enzymes with either glycogen or membranous structures (Drochmans, 1964, Wanson and Drochmans, 1968 a, b), and we chose the glycogen-sarcovesicular fraction isolated from skeletal muscle to investigate the gIycogen-enzyme-membrane interplay The present paper deals with the enzyme-glycogen complexes and their possible association with membrane-bound structures observed