scholarly journals 3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses.

1986 ◽  
Vol 103 (3) ◽  
pp. 875-886 ◽  
Author(s):  
G A Keller ◽  
M Pazirandeh ◽  
S Krisans
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Specific Activity ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Normal Liver ◽  
Hmg Coa Reductase ◽  
The Matrix ◽  
Coa Reductase ◽  
Liver Peroxisomes

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme involved in cholesterol biosynthesis, has recently been reported to be present in rat liver peroxisomes (Keller, G.A., M.C. Barton, D.J. Shapiro, and S.J. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:770-774). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is present in the matrix of peroxisomes. This study is a quantitative biochemical and immunoelectron microscopical analysis of HMG-CoA reductase in rat liver peroxisomes and microsomes of normal and cholestyramine-treated animals. Cholestyramine treatment produced a six- to sevenfold increase in the specific activity of peroxisomal HMG-CoA reductase, whereas the microsomal HMG-CoA reductase specific activity increased by about twofold. Using a computer program that calculates optimal linear combinations of marker enzymes, it was determined that between 20 and 30% of the total reductase activity was located in the peroxisomes of cholestyramine-treated animals. Less than 5% of the reductase activity was present in peroxisomes under control conditions. Quantitation of the immunoelectron microscopical data was in excellent agreement with the biochemical results. After cholestyramine treatment there was an eightfold increase in the density of gold particles per peroxisome, and we estimate about a threefold increase in the labeling of the ER.

scholarly journals The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects

1984 ◽  
Vol 219 (2) ◽  
pp. 461-470 ◽  
Author(s):  
D D Patel ◽  
C R Pullinger ◽  
B L Knight
Keyword(s):  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Specific Radioactivity ◽  
Density Lipoprotein ◽  
Cellular Protein ◽  
True Rate ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
In Cells

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.

scholarly journals A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme

1977 ◽  
Vol 164 (3) ◽  
pp. 501-508 ◽  
Author(s):  
Y A Baqir ◽  
R Booth
Keyword(s):  
Rat Liver ◽  
Cholesterol Ester ◽  
Time Course ◽  
Coenzyme A ◽  
Reductase Activity ◽  
Dietary Cholesterol ◽  
New Method ◽  
Coa Reductase ◽  
Microsomal Cholesterol ◽  
Coenzyme A Reductase

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.

Dose-Dependent Effect of Hydroxymethylglutaryl – Coenzyme A Reductase Inhibitor on Serum Cholesterol with Limited Dietary Restrictions: A Case Study

1993 ◽  
Vol 21 (2) ◽  
pp. 105-111
Author(s):  
S Okada ◽  
K Ichiki ◽  
S Tanokuchi ◽  
Z Ota
Keyword(s):  
Serum Cholesterol ◽  
Reductase Inhibitor ◽  
Coenzyme A ◽  
Cholesterol Biosynthesis ◽  
Dependent Diabetes Mellitus ◽  
Hmg Coa Reductase ◽  
Dietary Restrictions ◽  
Hmg Coa Reductase Inhibitor ◽  
Coa Reductase ◽  
Dose Dependent

Hydroxymethylglutaryl–coenzyme A (HMG–CoA) reductase inhibitor (pravastatin sodium) can selectively inhibit cholesterol biosynthesis in the liver and may lower serum cholesterol concentrations even where there are no particular dietary restrictions. A 72-year old housewife with non-insulin-dependent diabetes mellitus complicated by hyperlipaemia type IIb, who did not follow directions for diet therapy or kinesitherapy, was administered HMG–CoA reductase inhibitor. The initial dose of 10 mg/day HMG–CoA reductase inhibitor was increased by 10 mg/day every 4 weeks to 30 mg/day, maintained at 30 mg/day for 8 weeks and then reduced gradually until discontinuation after a further 27 weeks. Test results showed the changes in low-density lipoprotein cholesterol and apoprotein B to be dose-dependent. The findings represent the first clinical evidence that hypercholesterolaemia can be adequately managed by the use of HMG–CoA reductase inhibitor, even when no specific dietary restrictions are imposed, and may contribute to improvements in the quality of daily life for many patients suffering from hyperlipaemia type IIb.

scholarly journals Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol

1989 ◽  
Vol 264 (2) ◽  
pp. 495-502 ◽  
Author(s):  
J Iglesias ◽  
G F Gibbons
Keyword(s):  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Subcellular Fractions ◽  
Hmg Coa Reductase ◽  
Hepatocyte Cultures ◽  
C 32 ◽  
Lanosterol Demethylase ◽  
Cholesterol Precursor ◽  
Coa Reductase

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.

scholarly journals Inhibition of hydroxymethylglutaryl coenzyme A reductase activity induces a paradoxical increase in DNA synthesis in myeloid leukemia cells

Blood ◽  
1991 ◽  
Vol 77 (5) ◽  
pp. 1064-1070 ◽  
Author(s):  
RJ Hohl ◽  
RA Larson ◽  
V Mannickarottu ◽  
S Yachnin
Keyword(s):  
Dna Synthesis ◽  
Myeloid Leukemia ◽  
Coenzyme A ◽  
Reductase Activity ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Hmg Coa Reductase ◽  
Cellular Dna ◽  
Coa Reductase ◽  
Inhibition Of Dna Synthesis

Abstract The effects of competitive inhibition of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase by compactin on the in vitro proliferation of peripheral blood myeloid leukemia cells were studied using the cells from 45 patients with acute myeloid leukemia or chronic myelogenous leukemia in blast phase. The cells from 58% of these patients showed a dose-related inhibition of DNA synthesis when incubated with compactin. Unexpectedly, cells from 18% of the patients were resistant to the inhibitory effects of compactin on DNA synthesis and responded to the HMG CoA reductase inhibition with an actual increase in the incorporation of 14C-labeled thymidine into DNA. Another 18% of the patients studied displayed both inhibition and stimulation of DNA synthesis in a biphasic response depending on the particular concentration of compactin used. The maximum enhanced rates of cellular DNA synthesis were observed with lower compactin concentrations (5 x 10(-7) mol/L) than were required for maximum inhibition of DNA synthesis (10(-5) mol/L). Leukemia cells displaying a stimulated response to compactin had a significantly lower baseline DNA synthetic rate than did cells that showed an inhibitory response of DNA synthesis to compactin. There was no correlation between these cells' varying DNA synthetic response to compactin and measures of baseline HMG CoA reductase activity or acetate conversion to cholesterol. Whereas the observation of cellular DNA synthesis stimulation by HMG CoA reductase inhibition has not been observed in other mammalian cells and seems paradoxical, explanations may emerge in light of our growing knowledge concerning the importance of isoprenylation for the function of certain cell regulatory proteins.

The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum

1994 ◽  
Vol 107 (9) ◽  
pp. 2635-2642
Author(s):  
L.W. Lecureux ◽  
B.W. Wattenberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Coenzyme A ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Dna Encoding ◽  
Hmg Coa Reductase ◽  
Steady State Levels ◽  
Coa Reductase ◽  
Membrane Anchoring ◽  
Beta Galactosidase

The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with beta-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990) J. Biol. Chem. 265, 22004–22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80–90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring beta-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in brains of adult and 7-day-old rats

1976 ◽  
Vol 154 (2) ◽  
pp. 559-560 ◽  
Author(s):  
M M. Sudjic ◽  
R Booth
Keyword(s):  
Rat Brain ◽  
Coenzyme A ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Adult Brain ◽  
Adult Rat ◽  
Adult Rat Brain ◽  
Old Rats ◽  
Coa Reductase ◽  
Coenzyme A Reductase

Rat brain contains 3-hydroxy-3-methylglutaryl-CoA reductase activity, but this enzyme is far more active in 7-day-old brain than in adult brain. This difference may partly explain why cholesterol biosynthesis is more rapid in growing than in adult rat brain.

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