Surface Particles of Sarcoplasmic Reticulum Membranes: STRUCTURAL, FEATURES OF THE ADENOSINE TRIPHOSPHATASE

Abstract

Abstract Particles 30 to 40 A in diameter are attached to the external surface of negatively stained membrane vesicles formed by the ATPase of sarcoplasmic reticulum. These particles are identical with the surface particles of isolated, intact sarcoplasmic reticulum first described by Ikemoto et al. (Ikemoto, N., Sreter, F. A., and Gergely, J. (1966) Fed. Proc. 25, 465). Tryptic digestion was used in an attempt to determine whether these particles were structural features of the ATPase protein. Treatment of sarcoplasmic reticulum with trypsin caused an initial activation but eventual decline of the ATPase activity to about one-fourth of the initial activity. Surface particles were missing only after the ATPase activity was reduced 3- to 4-fold. Protection of ATPase activity with sucrose or ATP during trypsin digestion also protected against loss of the surface particles. Trypsin digestion initially cleaved the ATPase protein to two peptides of molecular weights 45,000 and 55,000 without loss of the ATPase activity. These peptides did not represent native subunits of the ATPase since the molecular weight of the undigested ATPase was found to be 102,000 under a variety of conditions reported to break the molecule down to sub- units. An active ATPase containing only tryptic peptides could be isolated from digested sarcoplasmic reticulum. Continued digestion cleaved the ATPase to peptides of molecular weight about 10,000 and cleavage to these lower molecular weight peptides was correlated with loss of ATPase activity and loss of surface particles. Freeze fracture profiles of intact membranes revealed an asymmetric arrangement of 90- to 100-A globules in the hydrophobic interior of the membrane. The globules were still present in membranes from which the surface particles were removed by tryptic digestion and in which the ATPase was cleaved to very small peptides. These peptides were not solubilized by trypsin but remained membrane bound. We suggest that the ATPase protein consists of a hydrophobic globular portion embedded in the membrane and a hydrophilic extension, part of which is observed as a surface particle under conditions of negative staining.