Localization of Ca " + Mgt + - ATPase of the Sarcoplasmic Reticulum in Adult Rat Papillary Muscle ANNELISE O . JORGENSEN , AMY C

Localization of the Ca + Mgt+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively . The Ca + Mgt+ -ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria . This suggests that the Ca + Mgt+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca + Mgt+ -ATPase of the sarcolemma . These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle . In cardiac muscle the sarcoplasmic reticulum is the intracellular membrane system which, together with the transverse tubular system (T-system) and the sarcolemma, regulates the Ca" concentration of the myofibril and thereby the state of contraction and relaxation of the myocardium. Ultrastructural examination of cardiac muscle (9, 29, 32, 34) has shown that the sarcoplasmic reticulum forms a separate intracellular compartment in the muscle cells surrounding the myofibrils like a laced sleeve . The portion of the sarcoplasmic reticulum membrane that is located in close apposition to the T-system is called interior junctional sarcoplasmic reticulum membrane while that portion in close apposition to the sarcolemma is called peripheraljunctional sarcoplasmic reticulum membrane. The remainder of the membrane system is referred to as the free ornonjunctional sarcoplasmic reticulum membrane (34) . The mechanism for myofibril contraction in the cardiac muscle is similar to that in skeletal muscle . Contraction is induced when depolarization of the sarcolemma leads to an increase in the Ca" concentration of the myofibril (36); in turn, relaxation sets in when Cat' is removed from the myofibril . It is generally agreed that in mammalian cardiac muscle (33, 36) calcium is removed from the myofibril by active transport across the sarcoplasmic reticulum membrane to the lumen of the sarcoplasmic reticulum . However, the site of storage of Ca" during relaxation as well as the origin of Ca" required for the induction of contraction is a controversial issue . Cardiac muscle, in contrast to skeletal muscle, is very THE JOURNAL OF CELL BIOLOGY VOLUME 93 JUNE 1982 883-892 © The Rockefeller University Press 0021-9525/82/06/0883/10 $1 .00 sensitive to changes in extracellular Ca" concentration (23). Thus, although it is believed that some ofthe Ca" required for contraction is released from the lumen of the sarcoplasmic reticulum (8, 10), it is almost certain that some of the Ca" is also of extracellular origin (23). Accordingly, some Ca must be stored in the lumen of the sarcoplasmic reticulum during relaxation, while some must be transported to the extracellular space . So far it has not been possible to determine whether the sites where the sarcoplasmic reticulum membrane is in close apposition to the T-tubules and/or the sarcolemma are sites of excitation-contraction coupling and/or sites of Ca transport in and out of the muscle cells . To understand the possible specific functions of the morphologically specialized regions of the sarcoplasmic reticulum in the transport ofCat+ to and from the myofibril during the contraction-relaxation cycle, it is important to know the function of the sarcoplasmic reticulum proteins as well as the precise distribution of these proteins in the various regions of this membrane system. The presence of a Ca + Mgt'-dependent ATPase in mammalian cardiac sarcoplasmic reticulum (Ca + Mgt+-ATPase) has been reported and it is generally agreed that this protein, as in skeletal muscle, actively transports Ca from the myofibril to the lumen ofthe sarcoplasmic reticulum when relaxation sets in (36) . The biochemical characteristics ofthe protein from cardiac muscle are similar to those of the protein of skeletal muscle (4, 15, 25, 33, 44, 45) although the rate of Ca uptake by the cardiac protein is slower than the rate ofCa uptake by 883 on D ecem er 4, 2017 jcb.rress.org D ow nladed fom the skeletal protein (36). Several investigators have reported that there is also a Ca + Mgt+ ATPase in the sarcolemma of myocardial cells (3, 7, 14, 28, 35) . While some of the Ca" + Mgt+-ATPase activity reported to be associated with isolated sarcolemmal membrane vesicles might represent contamination with sarcoplasmic reticulum membrane vesicles, Caroni and Carafoli (2, 3) have. recently isolated a Ca + Mgt+dependent ATPase from purified sarcolemma and have shown that it is distinct from the sarcoplasmic reticulum enzyme in size and in that it is calmoduhn-dependent. To determine (a) whether the Ca + Mgt+-ATPase of the sarcoplasmic reticulum is confined to the sarcoplasmic reticulum or whether it is also present on the sarcolemmal membrane and (b) whether this Ca' + Mgt+-ATPase is uniformly distributed in the various regions of the sarcoplasmic reticulum membrane system, we have used affinity-purified antibodies against the Ca + Mgt+-ATPase from skeletal muscle sarcoplasmic reticulum to localize the Ca + Mg+2-ATPase in the adult rat papillary muscle by the indirect immunofluorescence and immunoferritin labeling techniques . We have found that the Ca + M82+-ATPase is confined to the sarcoplasmic reticulum in adult rat papillary muscle. Furthermore we have found that the Ca + Mgt+-ATPase is rather uniformly distributed within the nonjunctional region of the sarcoplasmic reticulum membrane but is apparently absent from the region where the sarcoplasmic reticulum membrane is located in close apposition to either the transverse tubules or the sarcolemma . MATERIALS AND METHODS Purification of Rat Ca 2+ + Mg 2+ -ATPase Rat skeletal muscle Cat' + Mg2.-ATPase was prepared as previously described for the purification of rabbit ATPase (27), except that the fractionation in ammonium acetate was tamed out at pH 8.35. The ATPase was separated from minor contaminating proteins by preparative SDSPAGE according to the procedure described by Laemmh (22) . The Cat' + Mgt+-ATPase protein band was then excised from the polyacrylamide gel and the protein eluted electrophoretically as previously described (20) . Isolation of the microsomal fraction from rat ventricular muscle enriched in sarcoplasmic reticulum wascarried out according to the procedure of Harigaya and Schwartz (11) as modified by Jones et al .