Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction.
NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.
D002852:Chromatography, Ion Exchange; D002934:Cinnamates / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013053:Spectrophotometry
D002934:Cinnamates; D005420:Flavoproteins; C029010:cinnamic acid; D009251:NADPH-Ferrihemoprotein Reductase