Konstantinos A. Mitropoulos

3-Hydroxy-3-methylglutaryl-coenzyme A reductase: The difference in the mechanism of the in vitro modulation by phosphorylation and dephosphorylation to modulation of enzyme activity by non-esterified cholesterol

Incubation of rat liver microsomal fraction in the presence of increasing concentration of a serum preparation and the re-isolation of the treated microsomal vesicles resulted in a progressive increase in the concentration of non-esterified cholesterol, a progressive decrease in the activity of hydroxymethylglutaryl-CoA reductase and progressive changes in the characteristics of the Arrhenius plots of the enzyme. The changes in the characteristics of the Arrhenius plots of the enzyme in the serum-treated preparations are consistent with a progressive increase in the concentration of non-esterified cholesterol in the environment of the hydroxymethylglutaryl-CoA reductase in endoplasmic reiticulum vesicles. The serum-treated preparations with high non-esterified cholesterol content showed a constant activation energy between 37 and 20 degrees C, whereas the enzyme in the non-treated microsomal fraction, the buffer-treated and the lipoprotein-deficient serum-treated preparations showed breaks in the activation energy at about 29 degrees C. The microsomal fraction from rats fed on the standard, cholesterol- or cholestyramine-supplemented diet showed considerable differences in the activity of hydroxymethylglutaryl-CoA reductase and differences in the characteristics of their Arrhenius plots. However, the incubation of the microsomal fraction from the rats in the three experimental conditions with ATP and Mg2+ and the further incubation of the inactivated enzyme with a preparation of cytosolic phosphoprotein phosphatase resulted in Arrhenius plots with similar characteristics to those of the corresponding original microsomal fraction. These results suggest that changes in the concentration of non-esterified cholesterol in the endoplasmic reticular membrane are responsible for the differences in the activity of hydroxymethylglutaryl-CoA reductase in the microsomal fraction from the rats in these dietary conditions.

The effect of interruption of the enterohepatic circulation of bile acids and of cholesterol feeding on cholesterol 7α-hydroxylase in relation to the diurnal rhythm in its activity

1. 1. Cholesterol 7α-hydroxylase activity was assayed by a direct method in liver microsomal preparations from rats adapted to controlled lighting and feeding and subjected to conditions known to modify hepatic synthesis of cholesterol and bile acids. The mass of microsomal cholesterol that acts as substrate for the enzyme (the substrate pool) was estimated in each set of conditions. 2. 2. In cholestyramine-fed and bile-fistula rats, the activity of the enzyme was increased, but the normal diurnal rhythm in activity was maintained. The size of the substrate pool was not increased in the treated animals. 3. 3. In bile-fistula rats the biliary excretion of bile acids and cholesterol assumed a diurnal rhythm, with a maximum at night and a minimum during the day. These results are discussed in relation to the diurnal rhythm in the activity of cholesterol 7α-hydroxylase and in the rate of synthesis of cholesterol in the liver. 4. 4. When cholesterol was added to the food, cholesterol 7α-hydroxylase activity was increased but enzyme activity continued to vary diurnally. Cholesterol feeding increased the size of the substrate pool, suggesting that the increase in activity may be due to an increase in the supply of substrate, rather than to increased capacity of the enzyme.

On the metchanism for the regulation of 3-hydroxy-3-methylglutaryl-coenzyme a reductase, of cholesterol 7α-hydroxylase and of acyl-coenzyme A: Cholesterol acyltransferase by free cholesterol

The administration of mevalonic acid to rats by intravenous injection resulted in a dose- and time-dependent increase in the activity of cholesterol 7alpha-hydroxylase in the liver microsomal fraction, a decrease in the microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and no significant change in the activity of acyl-coenzyme A:cholesterol acyltransferase or in the concentration of free and of esterified cholesterol in the liver microsomal fraction. However, the increased hepatic cholesterogenesis that follows the injection of mevalonic acid resulted in an increase of the size of the intracellular pool of cholesterol that is in the environment of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acts as substrate for cholesterol 7alpha-hydroxylase. The administration of mevalonic acid to rats by stomach tube resulted in an increase in the activity of cholesterol 7alpha-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase and in the concentration of cholesterol esters in the liver microsomal fraction, while there was a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

The influence of cholesterol on the activity, on the isothermic kinetics and on the temperature-induced kinetics of 3-hydroxy-3-methylglutaryl coenzyme a reductase.

Abstract The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was assayed in liver microsomal preparations from rats fed cholesterol, cholestyramine or the standard diet, without and after treatment with digitonin. The activity of the enzyme in the microsomal fraction was high in the preparations from rats fed cholestyramine and low in the preparations from rats fed cholesterol as compared with the activity of the enzyme in preparations from rats fed the standard diet. The concentration of free cholesterol and of cholesterol esters was different in the microsomal fraction from the rats in the three experimental conditions. Treatment of the microsomal fraction with digitonin resulted in all three cases in solubilization of free cholesterol and of cholesterol esters, but the specific activity of hydroxymethylglutaryl-CoA reductase was higher in the digitonin-treated preparation than in the corresponding microsomal fraction. There was no detectable activity of hydroxymethylglutaryl-CoA reductase in the supernatant obtained after treatment of the microsomal fraction with digitonin. Isothermic kinetics showed that hydroxymethylglutaryl-CoA reductase in the microsomal fraction from rats fed cholesterol has an affinity for substrate that is many times higher than that in the microsomal fraction from rats fed cholestyramine and that the enzyme in microsomes from rats fed the standard diet has an intermediate affinity for substrate. In all three experimental conditions enzyme activity in the microsomal fraction was inhibited at high substrate concentrations. Treatment of the microsomal fraction with digitonin has decreased the apparent Km, in all three conditions, as compared with the value observed with the corresponding untreated microsomes. In all three experimental conditions, preincubation of the enzyme present in the microsomal fraction or in digitonin-treated microsomal preparation, resulted in an increased V, but the apparent Km value obtained with the preincubated microsomal fraction was similar to the value obtained with the corresponding microsomal fraction assayed without preincubation. Preincubation revealed hydroxymethylglutaryl-CoA reductase activity in the supernatant obtained from the treatment of the microsomal fraction with digitonin, in all three experimental conditons. The soluble enzymes, from cholestyramine-fed rats and from rats fed the standard diet, had identical apparent Km values. The differences in the Arrhenius plots of hydroxymethylglutaryl-CoA reductase in the microsomal fractions from rats in the three experimental conditions and the changes brought about by the treatment of the various microsomal fractions with digitonin are consistent with the possibility that such differences are due to changes in the free cholesterol concentration in the membrane of the various microsomal preparations. It is suggested that there is a pool of free cholesterol in the immediate lipid environment of hydroxymethylglutaryl-CoA reductase and that the size of this pool is important for the intracellular regulation of hydroxymethylglutaryl-CoA reductase.

Diurnal variation in the activity of cholesterol 7α-hydroxylase in the livers of fed and fasted rats

The introduction of a hydroxyl group at the 7arposition of cholesterol is the first step in the major pathway for the conversion of cholesterol into cholic acid and other bile acids. Cholesterol 7a-hydroxylase, the enzyme catalyzing this step, is confined to the microsomal fraction of the liver cells and there is evidence to suggest that cytochrome P-450, together with an NADPH-dependent flavoprotein, are involved in this hydroxylation [ 11. Cholesterol 7ar-hydroxylase is probably an important rate-limiting enzyme in bile acid biosynthesis [2], and therefore the study of changes in the activity of this enzyme under different physiological conditions may be expected to provide information on the regulation of bile acid synthesis in liver. In view of this fact, it would be of interest to know whether the activity of cholesterol 7a-hydroxylase measured in vitro is sensitive to the nutritional, hormonal and other experimental conditions under which the experimental animals may be kept. In this communication it is shown that the activity of cholesterol 7cw-hydroxylase exhibits diurnal rhythmicity in the livers of both fed and fasted rats. The pattern of the cyclic rise and fall observed in the activity of this enzyme is similar to that of tyrosine transaminase measured in the soluble part of liver homogenates. The rise in the activity of cholesterol 7ar-hydroxylase is prevented by treating the rats with inhibitors of protein synthesis, such as cycioheximide or actinomycin D. A brief account of part of this work has been given elsewhere [3].

A possible role for cytochrome P-450 during the biosynthesis of zymosterol from lanosterol by Saccharomyces cerevisiae

Abstract A cell-free system has been obtained from Saccharomyces cerevisiae which is capable of efficiently converting lanosterol 1 to a mixture of 4-demethyl sterols, quantitatively the most important identifiable component of which was zymosterol. Little or no ergosterol was synthesized. In the presence of carbon monoxide, the rate of zymosterol biosynthesis from lanosterol was decreased by 57% compared with that observed in control incubations and the amount of unmetabolized lanosterol was greater. Mitochondrial electron transport inhibitors such as cyanide and antimycin A had no effect on the overall rate of 4-demethyl sterol biosynthesis from lanosterol nor on the degree of inhibition by carbon monoxide.

On the mechanism of regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase and of acyl coenzyme A: Cholesterol acyltransferase by dietary fat

The activity and the kinetic properties of hydroxymethylglutaryl-CoA reductase and of acyl-CoA:cholesterol acyltransferase in the liver microsomal fraction have been compared between rats fed on either unsaturated or on saturated fat. When rats wre fed for 12h on a compounded diet containing either safflower seed oil or tristearin the composition of the fatty acyl chains of the microsomal phospholipids was shown to be relatively more unsaturated in the rats that received the unsaturated fat. The activity of hydroxymethylglutaryl-CoA reductase in the microsomal fraction was considerably reduced in rats fed on compounded diet containing unsaturated fat whereas this dietary condition resulted in a considerable increase in the activity of acyl-CoA:cholesterol acyltransferase. Similar effects were observed after feeding rats for 12 h on a commercial diet supplemented with either safflower seed oil or with tristearin. The addition of 2% cholesterol to the fat-supplemented diets resulted in both cases in a decrease in hydroxymethylglutaryl-CoA reductase and an increse in acyl-CoA:cholesterol acyltransferase activity as compared with the corresponding values from the rats fed on the fat-supplemented diets with no cholesterol. The Arrhenius plots of hydroxymethylgutaryl-CoA reductase in the microsomal fraction from rats fed on fat-supplemented commercial diet for 12 h showed breaks in the activation energy at 29.6 degrees C for the preparations from rats fed on tristearin and 28 degrees C for those from rats fed on safflower seed oil. The activation energy of the enzyme was lower above and higher below the break for the preparations from rat fed on the unsaturated fat-supplemented diet. Similar differences were obtained from the comparison of the Arrhenius plots in the preparations from rats fed on saturated fat and those in the preparations from rats fed on unsaturated fat when the diet was compounded and given to the animals for 36 h. The addition of 2% cholesterol to the commercial diet supplemented with either saturated or unsaturated fat resulted in Arrhenius plots with a constant activation energy between 37 and 22 degrees C for the enzyme in microsomal preparations from both groups of rats. The apparent Km value for hydroxymethylglutaryl-CoA was lower for the reductase in microsomal preparations from rats fed on the unsaturated fat as compared with that for the enzyme in microsomal preparations from rats fed on saturated fat. There was also a decrease in the apparent Km value for oleic acid for the acyltransferase from rats fed on unsaturated fat as compared with that for the enzyme in the microsomal preparation from the rats fed on saturated fat. The results of the present study are consistent with higher concentration of free cholesterol in endoplasmic reticular membrane in the environment of the reductase and that of acyltransferase following the administration of dietary unsaturated fat as compared with that following the administation of saturated fat.

Lanosterol 14α-demethylase. Similarity of the enzyme system from yeast and rat liver

Abstract The chemical synthesis of 24,25-dihydro[32-14C]lanosterol is described. The incubation of this material with a cell-free system from Saccharomvoes cerevisiae or with a microsomal preparation from rat liver resulted in both cases in the release of [14C]formic acid. This result suggests that in the biosynthesis of ergosterol in yeast, as well as in that of cholesterol in higher animals, the 14α-methyl group of lanosterol is removed as formic acid. In both systems, the measurement of the rate of release of [14C]formic acid from 24,25-dihydro[32-14C]lanosterol provides a simple and direct assay of lanosterol 14α-demethylase. Carbon monoxide inhibited both yeast and liver 14α-demethylase.

The formation of lithocholic acid, chenodeoxycholic acid and other bile acids from 3β-hydroxychol-5-enoic acid in vitro and in vivo

Abstract Previous work has shown that 3β-hydroxycholenoic acid and several naturally occurring C 24 bile acids are formed from cholesterol incubated with rat-liver mitochondria. It is now shown that [ 3 H]3β-hydroxycholenoic acid is converted into lithocholic, chenodeoxycholic, 3α,6β-dihydroxycholanoic and β-muricholic acids in vitro . This suggests that 3β-hydroxycholenoic acid is an intermediate in the formation of C 24 bile acids from cholesterol in vitro . When [ 3 H]3β-hydroxycholenoic acid is injected intravenously into rats, 80% of the radioactivity is recovered in the bile within 2 h, mainly as lithocholic, chenodeoxycholic, 3α,6β-dihydroxycholanoic and β-muricholic acids. This raises the possibility that 3β-hydroxycholenoic acid lies on one of the pathways by which chenodeoxycholic acid is formed in vivo .

Effect of triarimol on cholesterol biosynthesis in rat-liver subcellular fractions

Abstract Cholesterol biosynthesis was studied in rat-liver subcellular fractions incubated with DL-[2- 14 C]mevalonic acid in the presence and absence of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidine methanol). Triarimol strongly inhibits incorporation of radioactivity into cholesterol and this results in a large accumulation of radioactive lanosterol and 24,25-dihydro-lanosterol. The inhibition of lanosterol 14α-demethylase by triarimol was confirmed by assay of the enzyme in rat-liver microsomal fraction in the presence and absence of the inhibitor. Apart from a slight inhibition of Δ 7 -sterol-Δ 5 -dehydrogenase, triarimol did not affect the activity of any other enzyme involved in cholesterol biosynthesis from acetate.