Document Detail

Microbial transformation of steroids--VII. Hydroxylation of progesterone by extracts of Phycomyces blakesleeanus.
MedLine Citation:
PMID:  2004046     Owner:  NLM     Status:  MEDLINE    
Post mitochondrial supernatants (S-12 extracts) were prepared from Phycomyces blakesleeanus by grinding washed and frozen mycelial cakes in fine sand and extracting the paste produced with buffer containing Tris-HCl pH 7.8 (0.1 M), EDTA (0.01 M), dithiothreitol (5 mM) and glycerol (10% v/v). The S-12 extracts, obtained in this way, reproducibly hydroxylated progesterone, producing 7 alpha- and 15 beta-hydroxyprogesterone the major products of whole-cell transformation. Cell-free progesterone hydroxylation was found to be approximately linearly dependent on extract concentration, to require reduced NADP (partly replaceable by NADH), and to be dependent on progesterone (apparent Km calculated to be 4 mM). K+ and Mg2+ were found not to be required. Maximum progesterone hydroxylation occurred after 2 h at pH 7.8 and at 24 degrees C. Using optimum conditions S-12 extracts were capable of hydroxylating between 5 and 15% of added progesterone (0.2 mM). Hydroxylation was found to be partially inhibited by carbon monoxide (ca 40%) and almost completely inhibited by azoles, ketoconazole and diconazole. The NADPH and molecular oxygen requirements were replaceable by NaIO4. These findings strongly suggest that hydroxylation was being catalyzed by cytochrome P-450. This was confirmed by preparing progesterone-hydroxylating microsomes and Triton N-101-solubilized microsome extracts, and by obtaining a dithionite-reduced carbon monoxide-difference absorption spectrum peak at 455 nm in the solubilized microsome extracts.
K E Smith; S A Latif; D N Kirk
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of steroid biochemistry and molecular biology     Volume:  38     ISSN:  0960-0760     ISO Abbreviation:  J. Steroid Biochem. Mol. Biol.     Publication Date:  1991 Feb 
Date Detail:
Created Date:  1991-04-22     Completed Date:  1991-04-22     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9015483     Medline TA:  J Steroid Biochem Mol Biol     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  249-56     Citation Subset:  IM    
School of Biological Sciences, Queen Mary and Westfield College, London, England.
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MeSH Terms
Carbon Monoxide / pharmacology
Cell Fractionation
Cytochrome P-450 Enzyme System / metabolism*
Dithionite / pharmacology
Hydroxyprogesterones / metabolism*
Microsomes / drug effects,  enzymology
NAD / metabolism
NADP / metabolism
Phycomyces / drug effects,  enzymology,  metabolism*
Steroid 11-beta-Hydroxylase / metabolism
Reg. No./Substance:
0/Hydroxyprogesterones; 14844-07-6/Dithionite; 27534-93-6/7-hydroxyprogesterone; 53-59-8/NADP; 53-84-9/NAD; 630-08-0/Carbon Monoxide; 9035-51-2/Cytochrome P-450 Enzyme System; EC 11-beta-Hydroxylase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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