Purification and properties of cytochrome P-450-dependent 14 alpha-sterol demethylase from Candida albicans.
The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described. Optimal purification (875-fold) was achieved by extracting the cytochrome from microsomes with sodium cholate followed by hydroxyapatite, octyl-Sepharose and CM-Sepharose chromatographies, giving a cytochrome preparation of 17.5 nmol/mg of protein. By the use of SDS/polyacrylamide-gel electrophoresis the cytochrome was judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein. The Mr of P-450DM was estimated to be 51,000. The absorption spectrum of oxidized P-450DM was characteristic of a low-spin cytochrome, and its reduced CO complex had a Soret absorption peak at 447 nm. When reconstituted in a model membrane system of dilauroylphosphatidylcholine with NADPH and O2, P-450DM catalysed the complete 14 alpha-demethylation of lanosterol, which was inhibited by CO. The cytochrome appeared to have a high degree of substrate specificity; it was unable to oxidize a number of xenobiotic compounds in the reconstituted assay.
Hitchcock
C A
CA
Department of Microbiology, University of Leeds, U.K.
Dickinson
K
K
Brown
S B
SB
Evans
E G
EG
Adams
D J
DJ
D002176:Candida albicans / Q000201:enzymology*; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System; D003576:Electron Transport Complex IV / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007810:Lanosterol / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D013053:Spectrophotometry; D058886:Sterol 14-Demethylase
D010713:Phosphatidylcholines; C015153:1,2-dilauroylphosphatidylcholine; D007810:Lanosterol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase; D003576:Electron Transport Complex IV