title: Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.
abstract: OBJECTIVE
  To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro.
  
  
  METHODS
  Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21.
  
  
  METHODS
  The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH.
  
  
  RESULTS
  Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21.
  
  
  CONCLUSIONS
  Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.
authors.affiliation: Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, 739-8521 Japan.
authors.forename: L
authors.initials: L
authors.lastname: Nikfarjam
authors.forename: S
authors.initials: S
authors.lastname: Kominami
authors.forename: T
authors.initials: T
authors.lastname: Yamazaki
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: Hewer
authors.forename: C Dal
authors.initials: CD
authors.lastname: Pra
authors.forename: T
authors.initials: T
authors.lastname: Nakamatsu
authors.forename: C
authors.initials: C
authors.lastname: Betterle
authors.forename: R
authors.initials: R
authors.lastname: Zanchetta
authors.forename: B Rees
authors.initials: BR
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Furmaniak
mesh_terms: D000224:Addison Disease / Q000201:enzymology* / Q000276:immunology; D001323:Autoantibodies / Q000378:metabolism*; D002248:Carbon Monoxide / Q000494:pharmacology; D004227:Dithionite; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013255:Steroid 21-Hydroxylase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D004227:Dithionite; D002248:Carbon Monoxide; D013255:Steroid 21-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase