Isolation of cytochrome P-450 components from marmoset liver microsomes by high-performance liquid chromatography.
A fast protein liquid chromatographic (FPLC) system with pre-packed and laboratory-packed columns was used for the analytical and preparative isolation of marmoset monkey cytochrome P-450 (P450) and NADPH-P450-reductase. Chromatographic separations also allowed the recovery of cytochrome b5, NADH-b5-reductase and epoxide hydratase. Cholate-solubilized liver microsomes from phenobarbital-induced marmosets were crudely purified on 8-aminooctyl-Sepharose or 6-aminohexyl-Sepharose and then fractionated into several isoenzyme groups using hydroxyapatite. Further purification on Mono S or CM-Sepharose and finally on phenyl-Superose, phenyl-Sepharose or octyl-Sepharose yielded a P450 fraction which was apparently homogeneous as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the automated Phast system using silver staining. Removal of excess of non-ionic detergent was effected by hydroxyapatite columns, and this was compared with other methods. For the isolation of P450 isoenzymes from untreated marmosets, Mono Q columns were employed and yielded at least two highly purified forms. NADPH-P450-reductase was recovered from the 8-aminooctyl-Sepharose column or crudely fractionated on DEAE-Sepharose Fast Flow. Subsequent purification via 2',5'-ADP-Sepharose and Superose 12 chromatography resulted in a homogeneous preparation.
Kastner
M
M
Institut für Toxikologie und Embryopharmakologie, Berlin, Germany.
Neubert
D
D
D000818:Animals; D002144:Callithrix; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D003579:Cytochrome Reductases / Q000302:isolation & purification; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000302:isolation & purification; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification*; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification*
D003902:Detergents; D007527:Isoenzymes; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D009251:NADPH-Ferrihemoprotein Reductase