ESCHERICHIA-COLI FLAVODOXIN SEPHAROSE AS AN AFFINITY RESIN FOR CYTOCHROMES P450 AND USE TO IDENTIFY A PUTATIVE CYTOCHROME P450C17 3-BETA-HYDROXYSTEROID DEHYDROGENASE INTERACTION
Cm. Jenkins et al., ESCHERICHIA-COLI FLAVODOXIN SEPHAROSE AS AN AFFINITY RESIN FOR CYTOCHROMES P450 AND USE TO IDENTIFY A PUTATIVE CYTOCHROME P450C17 3-BETA-HYDROXYSTEROID DEHYDROGENASE INTERACTION, Archives of biochemistry and biophysics, 347(1), 1997, pp. 93-102
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.