CHARACTERIZATION OF PLASMA MEMBRANE OF MYCOPLASMA LAIDLAWII .V. EFFECTS OF SELECTIVE REMOVAL OF PROTEIN AND LIPID

Purified plasma membranes of Mycoplasma laidlawii were subjected to proteolytic enzyme digestion and aqueous acetone extraction. Up to 80% membrane protein could be removed by pronase, with a concomitant decrease in density of residual banding material. This membrane residue was still organized in vesicles and showed a decreased thickness and contrast in electron micrographs. Despite degradation of all characteristic membrane proteins (as shown by detergent-polyacrylamide gel electrophoresis) some protein fragments were retained in vesicular residues. The amino acid composition of this material was indistinguishable from that of total membrane protein, arguing against any enrichment in hydrophobic protein regions. Membrane hexosamine was not solubilized by pronase or aqueous acetone extraction, and could be substantially purified by combining these techniques. Extraction with 90% aqueous acetone removed over 95% of labeled membrane lipid but left sedimentable, protein-rich membranous material with unit membrane morphology. Such lipid-depleted membranes banded in sucrose gradients at a density of 1.25 g/cm3 as a single narrow band, in contrast to the isopycnic density of 1.17-1.18 g/cm3 for native membranes. At low pronase concentrations, electrophoresis experiments suggested preferential pronase attack on some higher molecular weight membrane proteins; these same electrophoretic components were preferentially released at very low concentrations of sodium dodecyl sulfate,, indicating differences in the organization of proteins within the membrane. Analysis of membranes labeled with [14C]glucosamine or [14C]oleate by detergent-gel electrophoresis followed by gel fractionation and counting showed that all oleate-containing lipid moved as a single broad band, while glucosamine migrated as two bands, one extremely broad and heterogeneous. © 1969.