Membrane Formation by the Adenosine Triphosphatase of Sarcoplasmic Reticulum

Abstract

Abstract The purified ATPase of sarcoplasmic reticulum exists as particles with minimum dimensions of about 90 A when dispersed by deoxycholate. When the dispersing agent is removed, the enzyme spontaneously forms vesicular membranes. The membranes are 60 A in thickness when examined in thin section and show a trilaminar staining pattern. Freeze-fracture profiles of the membranes formed from the ATPase demonstrate the presence of globular subunits possibly corresponding to the ATPase molecules. The reformed membranes can be disrupted by treatment with trypsin but not by treatment with phospholipase C. In the latter case, the vesicles take on a signet ring appearance. The reformed membranes are free of the 30- to 50-A projecting particles which are characteristic of the sarcoplasmic reticulum membrane. It is concluded that the ATPase enzyme is a functional subunit and is likely to be a structural subunit of the sarcoplasmic reticulum membrane. The membranes formed by the enzyme may be one enzyme particle in thickness. The projecting particles, which are missing from the purified ATPase preparation, cannot be concerned with the hydrolysis of ATP nor with the site of calcium activation of the ATPase. Amino acid and phospholipid analyses of the purified enzyme are presented. The phospholipid and neutral lipid content of the ATPase is identical with that of sarcoplasmic reticulum. The molecular weight of the protein is determined to be 102,000 daltons. Phosphorylation of the enzyme with ATP-γ-32P has led to a maximal measured specific phosphoryl content of 7.1 nmoles per mg of protein or about 0.73 phosphoryl groups/102,000 daltons. Each molecule of 102,000 appears, therefore, to contain one active site.