title: The interaction of cytochrome P450 17alpha with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry.
abstract: The lysine residues of guinea pig P450 17alpha were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17alpha prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17alpha was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17alpha in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17alpha with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima 739-8528, Japan.
authors.forename: Laleh
authors.initials: L
authors.lastname: Nikfarjam
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Izumi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Shiro
authors.initials: S
authors.lastname: Kominami
mesh_terms: D000818:Animals; D006168:Guinea Pigs; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry*; D013696:Temperature
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D008239:Lysine