Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells.
NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2',5' ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with Km values of 24 microM for NADPH and 16 microM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min-1 nmol-1 P-450 protein and with a purified rabbit P-4502C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min-1 nmol-1 P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.
Imaishi
H
H
Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University, Japan.
Yamada
T
T
Ohkawa
H
H
D000632:Aminopyrine / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002934:Cinnamates / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010947:Plants, Toxic; D014026:Nicotiana / Q000201:enzymology* / Q000648:ultrastructure
D002934:Cinnamates; D003574:Cytochrome c Group; D000632:Aminopyrine; C029010:cinnamic acid; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase