PubMed Health⌕ Search

Biomedical subjects

M Sinensky

Publications and source records attributed to M Sinensky.

At least 55 records · Page 3Linked to original sources
PubMed1985

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis by a non-sterol mevalonate-derived product in Mev-1 cells. Apparent translational control.

A somatic cell mutant (Mev-1) auxotrophic for mevalonate by virtue of a complete lack of detectable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activity has been shown to demonstrate a requirement for a non-sterol mevalonate-derived product for regulation of synthesis of HMG-CoA reductase. A comparison of the effects of 25-hydroxycholesterol and the combination of 25-hydroxycholesterol and mevalonate on HMG-CoA reductase activity, synthesis, and mRNA levels in Mev-1 is presented in this report. The results show a close correlation between activity, rate of synthesis, and mRNA levels for Mev-1 cells treated with 25-hydroxycholesterol alone. Under the conditions of these experiments these effects are relatively small (approximately a 4-fold decrease). A much larger inhibition of HMG-CoA reductase activity and rate of synthesis (approximately 50-fold) is observed upon treatment of Mev-1 cells with a combination of 25-hydroxycholesterol and mevalonate. Yet, under these conditions mRNA levels are still reduced by only a factor of 4. These results are interpreted to suggest that the non-sterol mevalonate-derived regulatory product of HMG-CoA reductase acts by a translational control mechanism.

Animals↗
PubMed1985

A somatic cell mutant with a kinetically altered 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A somatic cell mutant has been isolated which is resistant to killing and growth inhibition by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The resistance phenotype is dominant with respect to the wild-type cell and can largely be ascribed to a 6-7-fold lowering of the KM for HMG-CoA. We thus conclude that mevinolin resistance can be utilized to obtain a genetic marker for the structural gene encoding HMH-CoA reductase.

Animals↗
PubMed1985

Defective macromolecule biosynthesis and cell-cycle progression in a mammalian cell starved for mevalonate.

The isolation of a somatic cell mutant (Mev-1) with a block in one of the mevalonate-biosynthesizing enzymes (3-hydroxy-3-methylglutaryl-coenzyme A synthase, EC 4.1.3.5) has afforded us the opportunity to test and to extend the hypothesis that a product of mevalonate biosynthesis other than cholesterol is required for cellular proliferation. We present evidence here that both DNA synthesis and protein synthesis are inhibited in this mutant by mevalonate starvation, although RNA synthesis appears to be unaffected. The loss of DNA synthesis and the loss of protein synthesis in this mutant appear to be due to independent processes. DNA synthesis is reversibly inhibited by mevalonate starvation at a unique point in the cell cycle. Resumption of DNA synthesis after readdition of mevalonate exhibits a long lag; the peak of S-phase DNA synthesis occurs approximately 17 hr after mevalonate readdition, suggesting that mevalonate starvation puts cells into a quiescent (G0) state owing to their failure to transit a restriction point. The loss of DNA biosynthesis in the Mev-1 cell is well correlated with the rate of turnover of mevalonate label of certain terpenylated polypeptides.

Adenine↗
PubMed1983

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts.

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.

Animals↗
PubMed1982

Analysis of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant auxotrophic for mevalonate.

Regulation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and synthesis by 25-hydroxycholesterol is aberrant in a somatic cell mutant of the Chinese hamster ovary K1 cell auxotrophic for mevalonate by virtue of a defect in HMG-CoA synthase activity. Normal regulation of HMG-CoA reductase activity and synthesis in this mutant by 25-hydroxycholesterol can be restored by simultaneous incubation with a small (0.4 mM) mevalonate supplement. Normal regulation of HMG-CoA reductase is also observed in a revertant of the mutant cell with normal HMG-CoA synthase activity.

Animals↗
PubMed1982

Isolation and characterization of a mammalian cell mutant defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 cell auxotrophic for mevalonic acid has been isolated by means of the bromodeoxyuridine-visible light technique. This mutant can incorporate labeled mevalonate but not labeled acetate into cholesterol and, thus, is apparently defective in mevalonate biosynthesis. The mutant is recessive with respect to the parental cell phenotype. Assessment of the in vitro activities of the enzymes responsible for mevalonate biosynthesis under varying growth conditions indicates that the mutant, Mev-1, is defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.

Animals↗
PubMed1982

Complementary recessive 25-hydroxycholesterol-resistant somatic cell mutants--assay of 25-hydroxycholesterol binding activity.

Complementation analysis of recessive 25-hydroxycholesterol-resistant mutants of the CHO-Kl cell shows the existence of at least two complementation groups, one of which is missing a binding activity for 25-hydroxycholesterol. Both complementation groups are shown to be refractory to inhibition of cellular HMG-CoA reductase activity and in the inhibition of biosynthesis of this enzyme by 25-hydroxycholesterol.

Animals↗
PubMed1981

Radioimmune precipitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Chinese hamster fibroblasts. Effect of 25-hydroxycholesterol.

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.

Animals↗
PubMed1981

The effect of reagents that increase membrane fluidity on the activity of 3-hydroxyl-3-methyl glutaryl coenzyme A reductase in the CHO-K1 cell.

The compounds cetyl trimethyl ammonium bromide (CTAB) and ethanol both decrease the order parameter of a spin probe embedded in cholesterol-lecithin liposomes, but CTAB produces lowering of the order parameter comparable to that produced by ethanol at a 10,000-fold lower concentration. Treatment of CHO-K1 cells with CTAB or ethanol at concentrations that produce comparable increases of membrane fluidity produce to 2- to 3-fold increase of microsomal membrane cholesterol to phospholipid ratio and a 2- to 3-fold increase of the activity of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Cells treated with CTAB or ethanol show a progressively decreasing capacity to accumulate alpha-aminoisobutyric acid with increasing drug treatment, but cells pre-treated with CTAB are relatively resistant to the effects of CTAB on alpha-aminoisobutyrate transport. The increase in HMG-CoA reductase by CTAB or ethanol is not observed when these compounds are added directly to cell extracts but, rather, is only observed after 8 hours or exposure of intact cells to these drugs. Actinomycin D and cycloheximide treatment prevent the increase in enzyme activity, and the increase is also blocked in a regulatory mutant of the CHO-K1 cell with permanently repressed HMG-CoA reductase activity. These data are consistent with a homeoviscous adaptation mechanism in the CHO-K1 cell, in which increased activity of HMG-CoA reductase, through a process requiring RNA and protein synthesis, compensates for conditions that increase membrane fluidity by increased cellular cholesterol biosynthesis and cholesterol to phospholipid ratio.

Aminoisobutyric Acids↗
PubMed1980

Somatic cell genetic analysis of regulation of expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Three new types of regulatory somatic cell mutants defective in expression of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase are described. They include a recessive mutant with abnormally high enzyme activity, apparently defective in degradation of the enzyme, and one, phenotypically a mevalonate auxotroph, that maintains permanently repressed levels of enzyme activity and are recessive, constitutive mutants. These mutants offer means for analyses of the mechanism of regulation of expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured fibroblasts.

Animals↗
PubMed1980

Adaptive alteration in phospholipid composition of plasma membranes from a somatic cell mutant defective in the regulation of cholesterol biosynthesis.

A somatic cell mutant (CR1) of a Chinese hamster ovary cell (CHO-K1) which has previously been shown to be defective in the regulation of cholesterol biosynthesis accumulates more cholesterol than the parental cell line in plasma membranes. Although such an increase in membrane cholesterol should lead to an increase in the order parameter of these membranes, as measured with an electron spin resonance spin probe, the order parameters of mutant and wild-type plasma membranes are identical--apparently because of an adaptive alteration in membrane phospholipid composition. The phospholipid compositions of mutant and wild-type cell plasma membranes are compared and the mutant is shown to have a threefold higher level of oleic acid and a twofold lower level of phosphatidylethanolamine than the wild type. These results are consistent with model studies which show that these compositional changes lead to lower-order parameters for phospholipid dispersions.

Adaptation, Physiological↗
PubMed1979

86Rb+ fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content.

Steady-state fluxes of 86Rb+ (as a tracer for K+) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247--1249). Analogous to previous findings in ascites tumor cells, 86Rb+ influx in the parent strain was differentiated into a ouabain-inhibitable 'pump' flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual 'leak' flux. On the basis of this flux characterization, 86Rb+ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, 86Rb+ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K+ in the steady state is independent of the ordering of membrane lipid acyl chains.

Animals↗
PubMed1979

Defective regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant.

A somatic cell mutant of the CHO-K1 cell selected to be resistant to the killing effects of 25-hydroxycholesterol in the absence of cholesterol is shown to be defective in the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity by 25-hydroxycholesterol, cholesterol, and lipoproteins, thus maintaining the enzyme activity found in cells in the absence of exogenous sterol constitutively. The mutants phenotype is shown to be dominant with respect to the wild type. Actinomycin D and cycloheximide prevent the increase of HMG-CoA reductase activity that occurs in the CHO-K1 cell when cholesterol is removed from medium. Degradation of the enzyme, measured during inhibition of protein synthesis by cycloheximide, occurs at the same rate in the mutant as in the wild type. Kinetic studies indicate that the Km for two substrates, the activation energy, and a break in the Arrhenius plot are the same for HMG-CoA reductase determined in wild type and mutant cells. From these studies it is concluded that the mutant is defective in the regulation of synthesis of HMG-CoA reductase. Of the four processes which determine cellular cholesterol levels: biosynthesis, esterification, efflux, and uptake, only biosynthesis is altered, demonstrating that these processes are not co-ordinately controlled as has been suggested previously.

Animals↗