Differential centrifugation separates cardiac sarcolemmal and endosomal membranes from Langendorff-perfused rat hearts

The application of subcellular fractionation protocols developed in soft ti ssues to fibrous organs such as the heart is unsuitable given the substanti al differences in subcellular structure these tissues exhibit. The purpose of this study was to develop a simple method for the separation of sarcolem ma and endosomes from isolated Langendorff-perfused rat hearts. Hearts were , homogenized with either an Ultra-Turrax homogenizer or a hand-held glass tissue grinder. Quantitative immunoblots assessed the enrichment of the sar colemmal proteins caveolin 3 and the sodium potassium ATPase and the endoso mal proteins rab4 and GLUT4 in different membrane fractions. Application of homogenates to sucrose and Percoll density gradients failed to resolve mem branes differentially enriched in sarcolemmal or endosomal marker proteins, indicating little difference in density between the sarcolemma and endosom es. However, successive spins of homogenates from a handheld glass tissue g rinder successfully separated the endosomes from the sarcolemma, indicating differences in masses between the two membrane fractions. Approximately 70 % of total caveolin 3 and sodium potassium ATPase immunoreactivity was in m embrane pellets up to 20,000g and approximately 85% of rab4 and GLUT4 in pe llets from 20,000-100,000g. In addition, 86% of ouabain-sensitive ATPase ac tivity (sodium potassium ATPase activity) was in membrane pellets up to 20, 000g. Therefore, sarcolemmal membranes were pelleted up to 20,000g, and end osomal membranes between 20,000 and 100,000g. Regional ischemia (40 min) fo llowed by reperfusion (60 min) caused the translocation of GLUT4 (but not r ab4) from the endosomal membranes to the sarcolemma in the area of the hear t subjected to ischemia. (C) 2001 Academic Press.