CATALYTIC PROPERTIES OF CYTOCHROME P-450(SCC) PURIFIED FROM THE HUMANPLACENTA - COMPARISON TO BOVINE CYTOCHROME P-450(SCC)

Citation
Rc. Tuckey et Kj. Cameron, CATALYTIC PROPERTIES OF CYTOCHROME P-450(SCC) PURIFIED FROM THE HUMANPLACENTA - COMPARISON TO BOVINE CYTOCHROME P-450(SCC), Biochimica et biophysica acta, 1163(2), 1993, pp. 185-194
Citations number
30
Categorie Soggetti
Biophysics,Biology
ISSN journal
00063002
Volume
1163
Issue
2
Year of publication
1993
Pages
185 - 194
Database
ISI
SICI code
0006-3002(1993)1163:2<185:CPOCPP>2.0.ZU;2-J
Abstract
Cytochrome P-450scc was purified from the human placenta by extraction of mitochondria with cholate and Emulgen 911, chromatography on pheny l-Sepharose and DEAE-Sephacel, and ammonium sulphate fractionation. Th e catalytic properties of the purified human cytochrome P-450scc were analysed in Tween-20 micelles and compared to those of bovine adrenal cytochrome P-450scc analysed in the same system. Both enzymes had the same K(m) for cholesterol and were stimulated by cardiolipin when the cholesterol concentration was subsaturating. Examination of the rates of pregnenolone synthesis from 20alpha-hydroxycholesterol, 22R-hydroxy cholesterol and 20alpha,22R-dihydroxycholesterol by human and bovine c ytochromes P-450scc revealed that the first hydroxylation (22R positio n) was rate-limiting for both in Tween-20 micelles. The rate of the 22 R-hydroxylation was further decreased when a 20alpha-hydroxyl group wa s already present on the cholesterol side-chain. The second hydroxylat ion occurred at about the same rate as the third hydroxylation for bot h enzymes. The rate of side-chain cleavage of 25-hydroxycholesterol by human cytochrome P-450., in Tween-20 micelles was low, the highest ra te being about 1% of the V(max) for cholesterol. Substrate inhibition was seen with high concentrations of 25-hydrocycholesterol. Conversion of 25-hydroxycholesterol to pregnenolone was accompanied by a build-u p of products with intact side-chains, which were probably intermediat es of the reaction. Side-chain cleavage of 25-hydroxycholesterol by bo vine cytochrome P-450scc showed similar characteristics to the human e nzyme, except that the highest velocity observed was approx. 25% of th e V(max) for cholesterol. Rates of cleavage of 25-hydroxycholesterol b y both enzymes were higher in dioleoylphosphatidylcholine vesicles tha n in Tween-20, but were still well below the V(max) for cholesterol an d showed substrate inhibition. This study shows that there is close si milarity in catalytic properties between human and bovine cytochromes P-450scc which suggests that the active site of the cytochrome is high ly conserved.