Properties of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Solubilized from Rat Liver and Hepatoma (original) (raw)
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Proceedings of the National Academy of Sciences, 1976
Unlike the normal liver, numerous transplantable rodent and human hepatomas are unable to alter their rate of sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-GoA) reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] activity in response to a dietary cholesterol challenge. It has been suggested that this metabolic defect is linked to the process of malignant transformation. Hepatoma 7288C "lacks" feedback regulation of cholesterol synthesis when grown in vivo but expresses this regulatory property when grown in vitro (then called HTC). Therefore, it was used as a model system to answer whether an established hepatoma cell line that modulates its rate of cholesterol synthesis in vitro can express this property when grown in vivo, and whether cells reisolated from the tumor mass have the same regulatory phenotype as before transplantation. Our results show that long-term growth of hepatoma 7288C in tissue culture has not caused a biotransf...
Archives of Biochemistry and Biophysics, 1998
The level of gene expression at which dietary cholesterol exerts feedback regulation on hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated using young male Sprague-Dawley rats. Previous studies suggested that this regulation might be exerted posttranscriptionally. Thus, possible regulation at the levels of catalytic efficiency, protein turnover, and translation was investigated. To examine possible regulation at the level of catalytic efficiency, rats were placed on chow diets supplemented with 2% cholesterol and the rates of decline in hepatic HMG-CoA reductase activity and immunoreactive protein levels were determined. Both decreased slowly over a 72-h period. The catalytic efficiency did not change. These observations are inconsistent with phosphorylation-dephosphorylation or thiol-disulfide interchange as possible mechanisms. The possibility that dietary cholesterol might act by increasing the rate of turnover of HMG-CoA reductase protein was examined by determining the half-life of the enzyme in livers from rats consuming chow or chow supplemented with 2% cholesterol. The half-life of HMG-CoA reductase protein was not decreased in the animals receiving cholesterol, thus ruling out this possibility. Regulation at the level of translation was investigated by measuring the rate of HMG-CoA reductase protein synthesis in liver slices using [ 35 S]methionine and [ 35 S]cysteine. It was found that the rate of synthesis was reduced by over 80% in liver slices from rats fed a diet supplemented with 2% cholesterol. Similar results were obtained with liver slices from rats given mevalonolactone, which supplies both ste-rol and nonsterol endproducts. These results indicate that cholesterol regulates hepatic HMG-CoA reductase gene expression in rats primarily at the level of translation. . 2 Abbreviations used: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase; TCA, trichloroacetic acid; SRE, sterol response element; SREBP, sterol response element binding protein; PVDF, polyvinylidene fluoride; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; CHO, Chinese hamster ovary.
Biochemical Journal, 1989
Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.
Proceedings of the National Academy of Sciences of the United States of America, 1973
The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of hepatic cholesterol biosynthesis, is suppressed in human fibroblasts cultured in the presence of serum. This enzyme activity increases by more than 10-fold after the removal of serum from the medium. The rise in enzyme activity requires de novo protein synthesis and is not accompanied by changes in the activities of several other cellular enzymes. The factor responsible for the suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts is present in the sera of at least four mammalian species, and in human serum it is found in the low-density lipoproteins. Human highdensity lipoproteins, very low-density lipoproteins from chicken egg yolk, and the fraction of human serum containing no lipoproteins do not suppress the activity of 3hydroxy-3-methylglutaryl coenzyme A reductase.
Characteristics of3-hydroxy-3-methylglutaryl-CoA reductase from normal liver, Morris hepatomas 5123C, 5123t.c. and 9618A, and host liver were studied. Animals were fed on control and 5%-cholesterol diets. Microsomal membranes from all tissues were found to accumulate cholesterol after 3 days on the 5%-cholesterol diet. The enzyme of the tumours showed no feedback inhibition by dietary cholesterol, and that of host liver gave a variable response, whereas that of control liver was constantly inhibited by 90% or more. Arrhenius-plot analysis was conducted on the microsomal enzyme isolated from the various tissues. Control animals showed that the phase transition present at 270C was removed when animals were fed on 5%-cholesterol diet for 12h. The hepatomas failed to show this change even after 3 days of 5%-cholesterol diet and a significant increase in microsomal cholesterol. This failure to remove the break in Arrhenius plots also occurred in host liver, even though enzyme inhibition occurred. The reason why hepatomas fail to regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in response to dietary cholesterol interaction.
Regulation of cholesterol metabolism in a slow-growing hepatoma in vivo
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1988
Cholesterol metabolism and its regulation are altered in hepatomas as compared to normal liver. We investigated parameters of cholesterol metabolism and their regulation in rats bearing the well-differentiated Morris hepatoma 9108. The numbers of membrane associated receptors recognizing chylomicron remnants, the lipoproteins that deliver dietary lipid to the liver, were substantially decreased in the 910s tumor relative to the host liver. Cholesterol synthetic rates were 2-3-fold higher in the tumor, while the activity of 3-hydroxy3-methylglutarylcoenzyme A reductase (EC 1.1.1.88), a rate-limiting enzyme for sterol synthesis, was elevated 6-14-fold. Although tumor free and esterified cholesterol contents were elevated, the activity of acylcoenzyme Azcholesterol acyltransferase (EC 2.3.1X), the enzyme responsible for intracellular sterol esterification, was unchanged. Similar to the host liver, cholesterol synthesis and 3-hydroxy-3-methylglutarylcoenxyme A reductase were inhibited in the tumor when rats were fed a diet containing cholesterol, cholate and lard, and there was no effect on the numbers of chylomicron remnant recptors. Administering an intravenous bohts of very low density lipoproteins obtained from hypercholesterolemic rats caused an inhibition of tumor reductase activity, but had little effect on cholesterol content or cholesterol esterification. Thus, hepatoma 9108 expressed quantitative differences in cellular parameters involved in the uptake, metabolism, and synthesis of cholesterol and their susceptibility to regulation when compared with the host liver. These differences are best explained by changes in the hepatoma of multiple factors involved in the regulation of normal hepatic cholesterol metabolism.
The Journal of Lipid Research
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme catalyzing the rate-limiting step in cholesterol biosynthesis, exists in one active (dephosphorylated) and one inactive (phosphorylated) form in liver microsomes obtained from several animal species. T h e present study was undertaken in order to determine a ) whether the human enzyme also exists in active and inactive readily interconvertible forms; b) whether the large inter-individual variation in HMG-CoA reductase activity observed in normal man can be explained by variations in the activation state of the enzyme; and c) to characterize the reactivity of antibodies raised against rat liver HMG-CoA reductase with the intact human microsomal enzyme. HMG-CoA reductase activity, assayed in microsomes prepared in the presence of 50 mM NaF, was only 17 k 3% ' Even under standardized conditions' a wide range Of Ms. Lisbet Benthin, and the skillful preparation of the manustudy was supported by grants from the Swedish Medical (l2). The present study has demonstrated that this inter-
The Journal of Biological Chemistry, 1991
Feeding rats diets containing 2% cholesterol markedly reduced hepatic 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity but had little effect on mRNA levels. Addition of mevalonolactone to the diet further decreased reductase activity independent of a change in mRNA levels. In contrast, farnesyl pyrophosphate synthetase mRNA levels and enzyme activity were decreased to similar degrees in response to dietary cholesterol. Addition of mevalonolactone to the diet did not further decrease farnesyl pyrophosphate synthetase activity. Dietary cholesterol and mevalonolactone had no effect on mRNA levels for "cellular nucleic acid-binding protein" which has been demonstrated to bind the sterol regulatory elements in the HMG-CoA reductase and farnesyl pyrophosphate synthetase promoters. Dietary cholesterol increased cholesterol 7a-hydroxylase mRNA levels as expected. These results suggest that cholesterol-mediated feedback regulation of hepatic HMG-CoA reductase gene expression does not occur at the level of transcription.