A novel assay method is described for rapid quantitation of reaction rate of sterol Δ7-reductase (Δ 7-SR) which catalyzes reduction of the Δ7-double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-, Δ7-SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics (Km=210 μM, Vmax=1.93 nmol/min/mg) and inhibition of the rat hepatic Δ7-SR against well-studied cholesterol lowering agents such as triparanol (IC50=16 μM). 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) (IC50=5.2 μM), and trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) (IC50=0.25 μM). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., Δ7-SR, sterol Δ8-isomerase, and sterol Δ14-reductase), Δ7-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound (Ki=0.109 μM). Substrate specificity studies of the microsomal Δ7-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. Δ7-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin (≃5.0-fold). Finally, microsomal Δ7-SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammoniol-1-propanesulfonate (CHAPS) and enriched on PEG (0∼10%) precipitation, which should be suitable for further purification of the enzyme.
|Number of pages||8|
|Journal||Journal of biochemistry and molecular biology|
|Publication status||Published - 1997 Sep 30|
All Science Journal Classification (ASJC) codes
- Molecular Biology