AM1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50 degrees C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Measurement of human leukocyte microsomal HMG-CoA reductase activity

Methods were developed for determination of microsomal HMG-CoA reductase activity from freshly isolated human lymphocytes, monocytes, and granulocytes or cultured human lymphoid cells. Reductase activity in monocytes is approximately twice that in lymphocytes or granulocytes. The activity in cultured cells is approximately 34-fold greater than that in freshly isolated cells. Assay conditions were such as to preclude formation of HMG-CoA cleavage products. Leukocyte reductase activity was inhibited by dichloroacetate, a noncompetitive inhibitor of rat liver reductase and a serum cholesterol-lowering agent in man. Measurement of microsomal reductase activity from freshly isolated leukocytes may prove useful in assessing in vivo regulation of cholesterol synthesis in man.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration

Methods were developed for determination of human mononuclear leukocyte HMG-CoA reductase protein concentration by a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay procedure. Leukocyte microsomal HMG-CoA reductase, first immobilized onto a nitrocellulose filter, is sequentially reacted with 1) monospecific, polyclonal rabbit anti-rat liver HMG-CoA reductase antiserum, which crossreacts with the human liver and leukocyte enzymes; 2) biotinylated donkey anti-rabbit immunoglobulin; 3) a streptavidin-horseradish peroxidase conjugate; and 4) 4-chloro-1-naphthol and H2O2 to visualize the quantity of horseradish peroxidase bound to the immunocomplex. Color development was proportional to the quantity of either purified liver or leukocyte microsomal HMG-CoA reductase applied to the nitrocellulose. Color development was not observed, however, when HMG-CoA reductase was omitted from the nitrocellulose, when one of the reactant species was omitted from the incubation reactions, or when anti-rat liver HMG-CoA reductase antiserum was pre-absorbed with either rat liver or human leukocyte HMG-CoA reductase. Immunoreactivity of microsomal HMG-CoA reductase was independent of the phosphorylation state of the enzyme, but was inversely related to the concentration of thiol-reducing agents present in the microsomal preparation up to 4 mM. Further increases in thiol-reductant failed to produce changes in immunoreactivity. Freshly isolated mononuclear leukocyte microsomal HMG-CoA reductase protein concentration in leukocytes from 31 healthy, normocholesterolemic subjects was a linear function of HMG-CoA reductase activity (R = 0.65; P less than 0.001). The catalytic efficiency of the freshly isolated mononuclear leukocyte enzyme was 313 +/- 34 pmol of mevalonate formed per min of incubation at 37 degrees C per mg immunoreactive protein. This methodology, in conjunction with that recently developed to measure human leukocyte HMG-CoA reductase activity (1984. J. Lipid Res. 25: 967-978), should prove useful in discriminating between HMG-CoA reductase regulatory mechanisms involving changes in enzyme protein concentration and those resulting from changes in enzyme catalytic efficiency.     

Hydroxymethylglutaryl CoA reductase and the modulation of microsomal cholesterol content by the nonspecific lipid transfer protein

The influence of membrane cholesterol content on 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase, EC 1.1.1.34) in rat liver microsomes was investigated. Microsomes were enriched in cholesterol by incubation with egg phosphatidylcholine-cholesterol vesicles and the nonspecific lipid transfer protein from rat liver. By this method, the microsomal cholesterol content was 2.5-fold enhanced up to final concentrations of 140 nmol cholesterol per mg microsomal protein. In another experiment, microsomes isolated from rats fed a cholesterol-rich diet were depleted of cholesterol by incubation with egg phosphatidylcholine vesicles and the transfer protein. Both cholesterol enrichment and depletion had virtually no effect on the microsomal HMG-CoA reductase activity. In another set of experiments, normal rat liver microsomes were incubated with human serum, resulting in a rise of microsomal cholesterol content. This was reflected in an increase of acyl-CoA:cholesterol acyltransferase activity but failed to have an effect on HMG-CoA reductase.     

Regulation of cholesterol biosynthesis in sitosterolemia: effects of lovastatin, cholestyramine, and dietary sterol restriction.

We investigated the effects of lovastatin, cholestyramine, and dietary sterol restriction on cholesterol synthesis and low density lipoprotein receptor function in freshly isolated mononuclear leukocytes from two unrelated sitosterolemic families. Total plasma sterol concentrations were elevated in the two homozygous sitosterolemic subjects (343 and 301 vs. 185 mg/dl in controls) and contained increased amounts of plant sterols and 5 alpha-saturated stanols (20% and 8% vs. less than 1% in controls), but were not significantly different from controls in the two heterozygous subjects. The rates of conversion of acetate to cholesterol by mononuclear leukocytes were subnormal in all homozygous and heterozygous subjects and correlated with markedly reduced microsomal 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase activity. In the two homozygous subjects, cholestyramine treatment decreased plasma sterols 29% and 35%, and yet was associated with a paradoxical decline in mononuclear leukocyte HMG-CoA reductase activity. In contrast, plasma sterol concentrations decreased 14% and 5%, and mononuclear leukocyte HMG-CoA reductase activities increased 13% and 46% in three control and one heterozygous subjects treated with cholestyramine, respectively. Plasma sterol concentrations in the homozygous subjects unexpectedly failed to decline during treatment with lovastatin or a low sterol diet. In distinction, plasma sterol concentrations in three control and one heterozygous subjects dropped 28% and 31%, respectively, during treatment with lovastatin. Both cholestyramine and low dietary sterols stimulated low density lipoprotein receptor function. These results demonstrate a marked abnormality in cholesterol homeostasis in patients with homozygous sitosterolemia with xanthomatosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol-mediated regulation of HMG-CoA reductase in microsomes from human skin fibroblasts and rat liver

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was determined in microsomes from human skin fibroblasts and rat liver that had been variously manipulated in vivo or in tissue culture to up- and down-regulate the enzyme. The cholesterol content of these microsomal preparations was then altered by depletion to or enrichment from either cholesterol-free or cholesterol-rich lipid vesicles. Microsomes from human skin fibroblasts responded to cholesterol depletion by increasing HMG-CoA reductase activity and by decreasing it in response to cholesterol enrichment. This was independent of the initial enzyme activity or the tissue culture conditions. Alterations in cholesterol content of rat liver microsomes in vitro failed to demonstrate any significant changes in HMG-CoA reductase activity whether the microsomes started with low enzyme activity (cholesterol-fed rats) or with high enzyme activity (cholestyramine-treated rats). The results are discussed in relation to previously published data and in respect to differences in the control of the human skin fibroblast and rat liver enzymes.     

The pool of free cholesterol is not of major importance for regulation of the cholesterol 7 alpha-hydroxylase activity in rat liver microsomes

The relationship between the cholesterol 7 alpha-hydroxylase activity and the pool of free cholesterol in rat liver microsomes was studied under experimental conditions aimed to stimulate (biliary drainage, cholestyramine treatment, and lymphatic drainage) as well as inhibit (chenodeoxycholic acid treatment) bile acid synthesis. Highly accurate methods based on isotope dilution-mass spectrometry were used both for assay of the cholesterol 7 alpha-hydroxylase activity and the concentration of free cholesterol in the microsomes. In the assay of the cholesterol 7 alpha-hydroxylase, only endogenous cholesterol was used as substrate for the enzyme. Under the experimental conditions employed, the concentration of microsomal free cholesterol remained essentially unchanged in spite of a more than 20-fold variation in enzyme activity. It is concluded that the total pool of free cholesterol in the microsomes is not of major regulatory importance for the cholesterol 7 alpha-hydroxylase in rats.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

Investigations on the mechanism of the hypocholesterolemic action of 1-ethoxysilatrane

Summary Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 -hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation.A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbant assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.     

A cold-clamping technique for the rapid sampling of rat liver for studies on enzymes in separate cell fractions. Suitability for the study of enzymes regulated by reversible phosphorylation-dephosphorylation.

A technique for the rapid sampling, cooling and homogenization of rat liver is described. Its effectiveness in preserving the activity status of pyruvate kinase (soluble) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) (microsomal) during sampling is assessed in comparison with that of the freeze-clamping technique and of simple excision and mincing of liver tissue before homogenization. The results suggest that cold-clamping is equally effective as freeze-clamping in preserving the activity status of pyruvate kinase in liver samples obtained in situ, but in addition allows the subsequent separation of subcellular fractions, notably microsomes (microsomal fractions) and mitochondria. It is suggested that this property makes the technique useful in studying the activity status of enzymes (e.g. HMG-CoA reductase) the assay of which is subject to interference from the activity of other enzymes which are released from damaged organelles in crude homogenates of freeze-clamped liver samples. This suggestion was tested directly; the cold-clamping technique was found to preserve a substantially higher initial/total HMG-CoA reductase activity ratio [Easom & Zammit (1984) Biochem. J. 220, 739-745] in subsequently isolated microsomes compared with that obtained in microsomes prepared from liver samples processed in the conventional manner. The integrity of mitochondria isolated from homogenates of cold-clamped liver samples was preserved, as judged by the latency of intramitochondrial enzymes and by good respiratory control of the mitochondria. Possible further areas of metabolic studies to which the cold-clamping technique could be applied are suggested.     

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique.

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.     

Improved methods for the solubilization and assay of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A method for solubilizing HMG-CoA reductase is described that reproducibly yielded approximately 190% of the activity assayed in rat liver microsomes. Optimal solubilization occurred when microsomal membranes were frozen at a fixed concentration, thawed, homogenized in a buffer containing 50% glycerol, and incubated at 37 degrees C for 60 minutes. A rapid spectrophotometric assay of the reductase has been developed and the optimal conditions defined. Using this assay, the kinetics were determined for HMG-CoA reductase purified to a specific activity of 17,400 nmol NADPH oxidized per minute per mg protein.     

Influence of a naturally occurring competing enzymic activity on studies of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

An enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for D-hydroxymethylglutaryl CoA has been found in isolated rat liver microsomes and in microsomal extracts. The presence of this activity in enzyme preparations causes a decrease in the rate of mevalonate formation leading to an underestimation of reductase activity and an overestimation of the apparent Km of the reductase. The product formed by this competing enzymic activity behaves similarly to, but not identically with, mevalonolactone when chromatographed on Bio-Rad AG 1-x8 formate, which is used in many reductase assay procedures to separate mevalonolactone from hydroxymethylglutaryl CoA. Removal of this competing enzymic activity from reductase preparations can be accomplished by gel filtration using Bio-Gel A 1.5m, by washing the microsomes or by incubating the microsomal extract at 37 degrees C. Using enzyme preparations free of this competing enzymic activity, the apparent Km values of the reductase for D-hydroxymethylglutaryl CoA and NADPH were found to be 1.3 and 26 micronM respectively.     

Regulation of rat liver beta-hydroxy-beta-methylglutaryl coenzyme. A reductase activity and cholesterol levels of serum and liver in various dietary and hormonal states.

The effects of hormones and dietary factors on rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and serum and liver cholesterol levels were tested. Cholestyramine feeding markedly stimulated reductase activity in the livers of rats depleted of insulin or l-triiodothyronine. Therefore, these hormones are not absolute requirements for the stimulation of reductase activity.In hypophysectomized rats, l-triiodothyronine markedly stimulated reductase activity, even when the animals were cholesterol fed or fasted. However, this stimulation was accompanied by a reduction of serum and liver cholesterol levels. In diabetic rats, insulin failed to either stimulate reductase activity after cholesterol feeding, or to depress the level of liver cholesterol. These results are consistent with a model in which cholesterol functions as a feedback repressor of reductase activity.In contrast, a number of dietary and hormonal states produced little or no change in the level of serum and liver cholesterol while producing widely different reductase activities. These results suggest that the cholesterol level does not regulate reductase activity and cholesterol synthesis and that the factors that affect the formation of cholesterol also have a similar effect on its degradation. However, the possibility of a small subcellular pool of cholesterol regulating reductase activity and thus showing a positive correlation cannot be ruled out.The results reported in this paper suggest that the repressor, in a feedback repression model of regulation, should have similar effects on the rate-limiting enzymes of cholesterol synthesis and degradation. In this way a factor that operates through the repressor affects the rates of synthesis and degradation, but not the level of liver and serum cholesterol.     

Inactivation and activation of various membranal enzymes of the cholesterol biosynthetic pathway by digitonin

The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.     

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7 alpha-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7 alpha-hydroxylase and that substrate availability is of major regulatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7 alpha-hydroxylase. 1) The apparent degree of saturation of cholesterol 7 alpha-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7 alpha-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone.(ABSTRACT TRUNCATED AT 250 WORDS)