Preparation of a soluble 58kDa-3-hydroxy-3-methylglutaryl CoA reductase from liver microsomes and its inhibition by ethoxysilatrane, a hypocholesterolemic compound

Abstract

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58 kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50 kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58 kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50 kDa protein) to the membrane, present in the 58 kDa-protein appears to be involved in mediating the inhibition by silatrane.

Metal Complexes as Enzyme Inhibitors

“…Another silicon compound, ethoxysilatrane (Figure 24) has been shown to inhibit the 58k soluble 3-hydroxy-3-methylglutaryl (HMG) CoA reductase from liver microsomes. 200 Inhibition of HMG CoA reductase is a key step in the autoregulation of cholesterol biosynthesis, and HMG CoA reductase is the target for a number of antihyperlipidemic and antiatherosclerotic drugs. 201 The protein is usually membrane bound on the endoplasmic reticulum, and based on experiments with soluble and microsomal preparations of the protein, the site of interaction with inhibitors appears to be on the lumenal side.…”

“…Recent studies investigated a series of organosilicon compounds that inhibit diamine oxidase and semicarbazide-sensitive amine oxidase at micromolar K i . , Specificity remains to be proven, and a number of mechanisms for inhibition are proposed. Another silicon compound, ethoxysilatrane (Figure ) has been shown to inhibit the 58k soluble 3-hydroxy-3-methylglutaryl (HMG) CoA reductase from liver microsomes …”