Solubilization of pig lymphocyte plasma membrane and fractionation of some of the components.
The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23 degrees C. Some of the biological activities of the membrane survived this treatment. The leucine beta-naphthylamidase activity was more readily soluble than the 5'-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0 degrees C. Membrane solubilized in 2% deoxycholate at 23 degrees C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5'-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [(14)C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5'-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.
D000251:Adenosine Triphosphatases / Q000032:analysis; D000626:Aminopeptidases / Q000032:analysis; D000818:Animals; D001647:Bile Acids and Salts; D002462:Cell Membrane / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004589:Electrophoresis, Disc; D007930:Leucine; D008214:Lymphocytes / Q000032:analysis*; D008970:Molecular Weight; D009281:Naphthalenes; D009708:Nucleotidases / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012995:Solubility; D013552:Swine
D001647:Bile Acids and Salts; D009281:Naphthalenes; D010743:Phospholipids; D011506:Proteins; D009708:Nucleotidases; D000626:Aminopeptidases; D000251:Adenosine Triphosphatases; D007930:Leucine