Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Abstract

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of Sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875–885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electronopaque contents which serve as a Ca 2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the “standard” fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca 2+ since these channels are in the “open state” (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256–7259). Thus, the Ca 2+,Mg 2+-dependent ATPase (Ca 2+ ATPase) rate is only slightly enhanced in the presence of a Ca 2+ ionophore, which dissipates the Ca 2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca 2+ ATPase activity to Ca 2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca 2+ ATPase activity 4– to 7-fold. The Ca 2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca 2+ gradient has been generated, but can be stimulated 9– to 20-fold when the Ca 2+ gradient is dissipated with ionophore. This finding suggests that the Ca 2+ ATPase activity in SR is tightly coupled to Ca 2+ transport in situ.