Immobilized artificial membrane chromatography: rapid purification of functional membrane proteins.
A solid-phase membrane mimetic system, denoted as immobilized artificial membranes (IAM), has been developed and utilized as a novel high-performance liquid chromatography (HPLC) matrix for the first step in the rapid purification of functional membrane proteins. IAM phases consist of monolayers of amphiphilic membrane lipid molecules covalently bonded to a rigid silica particle. These monolayers of lipids have proved remarkably effective for the chromatography of biomolecules. Several cytochrome P450 isozymes, an extremely important family of hydrophobic membrane proteins with a labile heme catalytic center, have been partially purified in functional conformations from rat liver, kidney, and adrenal microsomes on IAM supports. Functionality of purified P450 and P450 reductase has been demonstrated by optical difference spectroscopy, by carbon monoxide binding, and by reconstitution of enzymatic activity in vitro. Other membrane proteins, including rat liver plasma membrane NADH oxidase and ferricyanide oxidoreductase have also been partially purified by IAM HPLC. The methods for purification of these proteins are described.
Pidgeon
C
C
Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907.
Stevens
J
J
Otto
S
S
Jefcoate
C
C
Marcus
C
C
D000311:Adrenal Glands / Q000201:enzymology; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D002845:Chromatography / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D003902:Detergents; D007668:Kidney / Q000201:enzymology; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification*; D008567:Membranes, Artificial; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D009994:Osmolar Concentration; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D003902:Detergents; D008565:Membrane Proteins; D008567:Membranes, Artificial; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital