The Plasma Membrane of Isolated Fat Cells: I. IDENTIFICATION OF TRYPSIN-SENSITIVE MEMBRANE PEPTIDES BY SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS

Abstract

Abstract Analysis of purified plasma membranes from isolated fat cells by sodium dodecyl sulfate polyacrylamide gel electrophoresis resolved at least 11 major peptide components which ranged in apparent molecular weights from 168,000 to 22,000. No alteration in the number or mobilities of these components was found when fat cells were isolated with three different preparations of partially characterized crude collagenase (Clostridium histolyticum) or if plasma membranes were purified on linear or discontinuous sucrose gradients. Fat cell plasma membranes contained two major glycopeptides with apparent molecular weights of 94,000 and 78,000. The isolated fat cell plasma membrane fraction consisted of approximately 40% protein, 40% phospholipid, 16% cholesterol, and 4% neutral carbohydrate. Essentially complete removal of phospholipid and cholesterol from membranes could be attained by extraction with ethanol at room temperature for 15 min. The alcohol-insoluble fraction consisted of about 95% protein, 4% neutral sugar, and 0.5% sialic acid. A diffuse Schiff reagent-positive band which migrated near the dye front on sodium dodecyl sulfate polyacrylamide gels was alcohol-soluble indicating the possible presence of glycolipid. The marked reduction in the ability of plasma membranes derived from trypsinized fat cells to bind 125I-insulin was accompanied by the selective digestion of two major membrane peptides of molecular weights 68,000 and 54,000 and the partial digestion of a 42,000 molecular weight peptide. The membrane glycoproteins were relatively resistant to trypsin treatment of intact cells or isolated plasma membranes in Krebs-Ringer phosphate buffer. In contrast, membranes suspended in hypotonic EDTA (1 mm) were uniformly degraded by trypsin, apparently as a consequence of altered conformations of the membrane or the membrane peptides, or both, in this medium.