Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.
Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2', 5'-adenosine diphosphate agarose. SDS-PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase.
Ponnamperuma
K
K
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340, USA.
Croteau
R
R
D000906:Antibodies; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
D000906:Antibodies; D009251:NADPH-Ferrihemoprotein Reductase