title: Application of high-pressure to subfractionate membrane protein-lipid complexes: a case study of protein kinase C.
abstract: Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.
authors.affiliation: Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: N
authors.initials: N
authors.lastname: Orr
authors.forename: E
authors.initials: E
authors.lastname: Yavin
authors.forename: M
authors.initials: M
authors.lastname: Shinitzky
authors.forename: D S
authors.initials: DS
authors.lastname: Lester
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D006874:Hydrostatic Pressure; D008297:Male; D008563:Membrane Lipids / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000302:isolation & purification*; D011493:Protein Kinase C / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
chemical_list: D008563:Membrane Lipids; D008565:Membrane Proteins; D011493:Protein Kinase C