Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100.
To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.
Wiedenmann
B
B
Lawley
K
K
Grund
C
C
Branton
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002417:Cattle; D003034:Coated Pits, Cell-Membrane / Q000032:analysis*; D011992:Endosomes / Q000032:analysis*; D006023:Glycoproteins / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D011189:Potassium Chloride / Q000494:pharmacology; D012995:Solubility
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011092:Polyethylene Glycols; D011189:Potassium Chloride