High-performance liquid chromatography assay for 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity in rat liver microsomes.
A quantitative HPLC method has been developed for measuring 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity in rat liver microsomes. Apparent Km values of 23 and 27 microM were estimated for 5-cholestene-3 beta,7 alpha-diol and NAD+, respectively. A temperature optimum of 45 degrees C was estimated using standard assay conditions. It was observed that feeding cholesterol (2%) or cholestyramine (5%) to rats increased (twofold) specific activity. This method should prove useful in studies of the regulation of bile acid synthesis and complement the HPLC assay technique recently developed for measuring cholesterol 7 alpha-hydroxylase activity.
Hylemon
P B
PB
Department of Medicine, Medical College of Virginia-VCU, Richmond 23298-0678.
Raynor
L
L
Bohdan
P M
PM
Vlahcevic
Z R
ZR
D015096:3-Hydroxysteroid Dehydrogenases / Q000032:analysis*; D000818:Animals; D002784:Cholesterol / Q000008:administration & dosage; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis; D002792:Cholestyramine Resin / Q000008:administration & dosage; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004789:Enzyme Activation; D006888:Hydroxycholesterols / Q000032:analysis; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009243:NAD / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D006888:Hydroxycholesterols; D009243:NAD; D002792:Cholestyramine Resin; D002784:Cholesterol; D015096:3-Hydroxysteroid Dehydrogenases; C031954:delta(5)-3 beta-hydroxysteroid dehydrogenase; D002790:Cholesterol 7-alpha-Hydroxylase