Abstract

A purified preparation of sarcoplasmic reticulum from rabbit skeletal muscle has been found to consist of a heterogeneous population of vesicles. Isopycnic centrifugation was used to obtain "light" and "heavy" vesicles from the upper and lower ends of a 25 to 45% (w/w) linear sucrose gradient. Each fraction accounted for about 10 to 15% of the total vesicles. The remainder of the vesicles were of intermediate density and banded between the light and heavy fraction. Light vesicles were composed of about equal amounts of phospholipid and Ca2+ pump protein which contained approx. 90% of the protein. Heavy vesicles contained in addition to the Ca2+ pump protein (55–65% of the protein) two other major protein components, the Ca2+ binding and M55 proteins which accounted for 20–25 and 5–7% of the protein of these vesicles, respectively. The sarcoplasmic reticulum subfractions had 32P-labelled phosphoenzyme levels proportional to their Ca2+ pump protein content and contained similar Ca2+ -stimulated ATPase activities. They were capable of accumulating Ca2+ in the presence of ATP and of releasing the accumulated Ca2+ when placed into a medium with a low Ca2+ concentration. The vesicles differed significantly in that heavy vesicles had a greater number of non-specific Ca2+ binding sites than light vesicles (approx. 220 vs 75 nmol of bound Ca2+ per mg protein), in accordance with their high content of Ca2+ binding protein. Electron dense material could be seen within the compartment of heavy but not light vesicles. Removal of Ca2+ binding and M55 proteins from heavy vesicles resulted in empty membranous structures consisting mainly of Ca2+ pump protein and phospholipid. Electron micrographs of sections of muscle showed dense material in terminal cisternae but not in longitudinal sections of sarcoplasmic reticulum. These experiments are consistent with the interpretation that (1) the electron dense material inside heavy vesicles may be referable to Ca2+ binding and/or M55 proteins, and that (2) light and heavy vesicles may be derived from the longitudinal sections and terminal cisternae of sarcoplasmic reticulum, respectively.