A novel assay for cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase activity using reversed-phase ion-pair chromatography: demonstration that Lifibrol (K12.148) modulates the enzyme activity.
Cytosolic HMG-CoA synthase and microsomal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyze two sequential steps in the mevalonate pathway. Both enzymes are negatively regulated by cholesterol. Cytosolic HMG-CoA synthase is responsible for the generation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA). We have developed a new method to determine HMG-CoA synthase activity. In this assay, HMG-CoA is formed from acetoacetyl-CoA and labeled acetyl-CoA. The HMG-CoA product is isolated from the reaction mixture by means of reversed-phase ion-pair chromatography. The recovery of the product was always greater than 90%. The average within-batch coefficient of variation for HMG-CoA synthase activity was 5.1%. Using the new assay, we demonstrate that Lifibrol (K12.148), a new hypolipidemic compound, inhibits HMG-CoA synthase. Because our assay is accurate and precise it may become useful in future studies on the regulation and the pharmacological modulation of cytosolic HMG-CoA synthase.
Scharnagl
H
H
Department of Internal Medicine (Division of Clinical Chemistry), Albert Ludwigs-University, Freiberg, Germany.
März
W
W
Schliack
M
M
Löser
R
R
Gross
W
W
D000818:Animals; D000440:Butanols / Q000494:pharmacology*; D002645:Chickens; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003600:Cytosol / Q000201:enzymology; D062385:Hydroxybenzoates / Q000494:pharmacology*; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000960:Hypolipidemic Agents / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology
D000440:Butanols; D062385:Hydroxybenzoates; D000960:Hypolipidemic Agents; C059343:lifibrol; D006904:Hydroxymethylglutaryl-CoA Synthase