Abstract
Antibody prepared against 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of rat liver can be shown to inhibit this enzyme in extracts prepared from cultured Chinese hamster ovary (CHO-K1) cells. The molecular weight (53,000) of the HMG-CoA reductase subunits of rat liver and Chinese hamster liver is identical with a [35S]methionine-labeled polypeptide that can be precipitated from CHO-K1 lysates by this antibody used in conjunction with protein A Sepharose. It is shown that 25-hydroxycholesterol which lowers HMG-CoA reductase activity in cultured fibroblasts blocks the incorporation of labeled methionine into this polypeptide. Furthermore, the antibody immune precipitates two other polypeptides with molecular weights of 127,000 and 60,000. The latter polypeptide responds to 25-hydroxycholesterol in the same fashion as the 53,000-dalton polypeptide. In a dominant 25-hydroxycholesterol-resistant mutant of the CHO-K1 cell, 25-hydroxycholesterol did not inhibit incorporation of labeled methionine into either the 53,000- or 60,000-dalton polypeptides.
Highlights
From theDivision of Cardiology, Department of Medicine, University of California, Los Angeles School of Medicine, Los Angeles, California 90024
Taryl coenzymeA (HMG-CoA) reductase of rat liver can hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)’ reducbe shown to inhibit this enzyme in extracts prepared tase, the enzyme responsible for the synthesis of mevalonic from cultured Chinese hamster ovary (CHO-K1) cells. acid and believed tobe the key regulatory enzyme in the
The mechanism by tase subunits of rat liverand Chinese hamster liver is which oxygenated sterols affect HMG-CoA reductase activity identicalwitha[36S]methionine-labeledpolypeptide in mammalian cells has not previously been directly demonthat can be precipitated from CHO-K1 lysates by this antibody usedin conjunction with proteiAn Sepharose
Summary
When cells are treatedwith 25-hydroxycholestero1,this compound is added in 10pl of ethanol to a final concentration of sterol of 0.5 pg/ ml. The beads were pelleted and resuspended in 100 pl of SDS sample buffer and heated at 100 OC for 5 min. The supernatant is 6.8,5% (v/v) 0-mercaptoethanol, and 0.001%bromphenol blue (SDS removed and mixed with specific antibody (0.1mg/ml final concen- sample buffer) and heated at 100 "C for 5 min before layering onto tration) and incubated with shaking for another hour at room tem- the gel. A slurry of proteinA beads in trichloroacetic acid and soaked in 1M sodium salicylate containphosphate-buffered saline (25 pl) was added and the samples ing 5%glycerol for 30 min with shaking. The enzyme was purified as has been mented with 2% delipidizedserum andthe cells incubated for another described for rat liver [10]to a final specific activity of 4.7 X lo3nmol. The cells were harvested by scraping into 600p1of0.05 M Tris, pH 7.4, in 0.15 M
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