European Journal of Endocrinology (2005) 152 95–101 ISSN 0804-4643 CLINICAL STUDY Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison’s disease L Nikfarjam1, S Kominami1, T Yamazaki1, S Chen2, R Hewer2, C Dal Pra2,3, T Nakamatsu2,3, C Betterle4, R Zanchetta4, B Rees Smith2,3 and J Furmaniak2,3 1Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, 739-8521 Japan, 2FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK, 3Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK and 4Department of Medical and Surgical Sciences, University of Padua, Via Ospedale Civile 105, 35128 Padua, Italy (Correspondence should be addressed to J Furmaniak, FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK; Email: fi
[email protected]) 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. Design: 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 dinu- cleotide 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 micro- somes in the presence of CO after addition of NADPH.