Michael Waterman
Faculty Member
Last active: 2/12/2015

Escherichia coli flavodoxin sepharose as an affinity resin for cytochromes P450 and use to identify a putative cytochrome P450c17/3beta-hydroxysteroid dehydrogenase interaction.

Jenkins CM, Pikuleva I, Kagawa N, Waterman MR
Arch Biochem Biophys. 1997 347 (1): 93-102

PMID: 9344469 · DOI:10.1006/abbi.1997.0318

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E. coli culture) recombinant E. coli Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation. Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3beta-HSD covalently linked 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 not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro. From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum.

Copyright 1997 Academic Press.

MeSH Terms (18)

3-Hydroxysteroid Dehydrogenases Adrenal Cortex Animals Cattle Chromatography, Affinity Cloning, Molecular Cytochrome P-450 Enzyme System Electrophoresis, Polyacrylamide Gel Escherichia coli Flavodoxin Immunoblotting Peptide Fragments Protein Binding Recombinant Proteins Sepharose Steroid 17-alpha-Hydroxylase Steroid 21-Hydroxylase Steroids

Connections (1)

This publication is referenced by other Labnodes entities:

Links