Isolation of sealed vesicles highly enriched with sarcolemma markers from canine ventricle

A highly enriched sarcolemma preparation was isolated by a combination of homogenizations and differential centrifugations of a homogenate of canine ventricular tissue followed by centrifugation of a membrane fraction layered over 24% (w/v) sucrose. The membrane fragments in the preparation were found to reside in a vesicular configuration by electron microscopy. Adenylate cyclase activity, specific ouabain binding sites, ouabain-sensitive potassium phosphatase activity and high-affinity (?)dihydroalprenolol binding sites were enriched from 27- to $\text{>40-}\text{fold}$ compared to the homogenate. 5′-Nucleotidase and antimycin A-insensitive NADH-cytochrome c reductase activities were enriched 10.1- and 3.0–3.9-fold, respectively, compared to the homogenate. Conversely, Ca²⁺-ATPase and succinic dehydrogenase activities were somewhat less than those expressed by the homogenate. The vesicles, or at least a fraction thereof, in the preparation were osmotically active as monitored by changes in 90° light scatter upon increases in the osmolarity of the extravesicular medium. $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity in the preparation was 23.6 and 99.9 μmol/mg per h before and after 15 consecutive freeze-thaw cycles, respectively. This suggests that the preparation contains a large fraction of intact right-side-out vesicles but that about 24% of the vesicles in the preparation may be freely permeable. Conversely, the number of specific ouabain binding sites was increased about 1.4-fold by the freeze-thaw cycles which is consistent with the presence of