ESCHERICHIA-COLI FLAVODOXIN SEPHAROSE AS AN AFFINITY RESIN FOR CYTOCHROMES P450 AND USE TO IDENTIFY A PUTATIVE CYTOCHROME P450C17 3-BETA-HYDROXYSTEROID DEHYDROGENASE INTERACTION

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed t o demonstrate an interaction between native Escherichia coil Fld and c ytochrome P450c17, has been synthesized, using highly expressed (7 mu mol Fld/liter E. coil culture) recombinant E. coil Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the speci ficity of Fld Sepharose, we have examined the utility of this resin fo r purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of t his mixture on Fld Sepharose resulted in a threefold enrichment of cyt ochrome P450 specific content without spectrally detectable P450 denat uration. Electrophoretic and immunoblot analyses of fractions eluted f rom the Fld Sepharose column revealed the presence of P450c17 and P450 c21, both of which were sufficiently pure, after SDS-PAGE, for identif ication by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3 beta-hydroxy steroid dehydrogenase (3 beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3 beta-HSD covalently li nked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen. This interaction, however, does no t appear to affect P450c17 hydroxylase and lyase activities as measure d in vitro. From these results, we propose that 3 beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the end oplasmic reticulum. (C) 1997 Academic Press.