Recovery and activation of hydroxymethylglutaryl coenzyme A reductase from rat small intestine.
We describe a method for estimating the total activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.34) in the small intestine of rats. An homogenate of the whole small intestine is prepared rapidly and assayed directly to maximize the yield of enzyme and to minimize opportunity for uncontrolled change in activity. Fresh homogenate inhibits the expression of reductase in hepatic microsomes, has high HMG-CoA cleavage activity, and forms NADPH-independent metabolites which contaminate mevalonolactone isolated by thin-layer chromatography. When homogenate is preincubated, these interfering factors are decreased and reductase activity is increased. Part of this increase can be inhibited by F-. After freezing and preincubation, total reductase activity recovered from homogenates of small intestine from 300-g male rats at the middle of their dark period is 40 nmol mevalonate per min compared to 70 from hepatic microsomes. If F- is added at the time of homogenization, an activity of 11 nmol per min is recovered from each organ. Reductase in intestinal homogenate has an apparent Km for S-HMG-CoA of 4 microM.
D000818:Animals; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D005260:Female; D005459:Fluorides / Q000494:pharmacology; D005615:Freezing; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007421:Intestine, Small / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology
D006903:Hydroxymethylglutaryl CoA Reductases; D005459:Fluorides