Purification and partial characterization of NADPH-cytochrome P450 reductase from Gentiana triflora flowers.
Reduced form of nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase was solubilized from a microsomal fraction of Gentiana triflora flowers by 3-[(3 Cholamidopropyl)-dimethylammonio]-1-propane sulfonate detergent and purified to electrophoretic homogeneity. The purification was achieved by adenosine 2', 5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. A Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. Western blot analysis showed that the purified protein cross-reacted with polyclonal antibody raised against rabbit anti-Gentiana triflora NADPH-cytochrome P450 reductase antibodies. The temperature and pH optimum for reduction of cytochrome c was 25 degrees C and 7.4 respectively. The Km values for the binding of NADPH and cytochrome c were 9.4 and 3.2 microM, respectively. In this paper, we present some results of the purification and partial characterization of microsomal NADPH-cytochrome P450 reductase from Gentiana triflora flowers.
Kim
M J
MJ
Department of Horticultural Science, Sangmyung University, Chonan, Korea.
Kim
Y
Y
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003116:Color; D003429:Cross Reactions; D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013696:Temperature
D003574:Cytochrome c Group; D007074:Immunoglobulin G; D010940:Plant Proteins; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase