부산대학교 도서관

A novel assay method is described for rapid quantitation of reaction rate of sterol Δ^7-reductase (Δ^7-SR) which catalyzes reduction of the Δ^7-double bond of sterols. Of six different organ tissues-liver. small intestine, brain, lung. kidney. and testis-.Δ^7-SR activity was detected only in liver (2.30 nmol/min ㎎ protein) and testis (0.11 nmol/min-㎎ protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate. we assessed both kinetics (K_m=210 μM, V_(max)=1.93 nmol/min/㎎) and inhibition of the rat hepatic Δ^7-SR against well-studied cholesterol lowering agents such as triparanol (IC_(50)=16 μM). 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) (IC_(50)=5.2 μM), and trans-l,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) (IC_(50)=0.25 μM). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e..Δ^7-SR, sterol Δ^8-isomerase. and sterol Δ^(14)-reductase). Δ^7-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound (K_j=0.109 μM). Substrate specificity studies of the microsomal Δ^7-SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. Δ^7-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol ($gt;2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin (◎5.0-told). Finally. microsomal Δ^7-SR was solubilized by 1.5% 3-[3(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0∼10%) precipitation, which should be suitable for further purification of the enzyme.