[Isoelectric focusing of 3-hydroxy-3-methylglutaryl-CoA-reductase from the postmicrosomal fraction of rat liver]. / Izoélektrofokusirovanie 3-gidroksi-3-metilglutaril-CoA-redutazy postmikrosomnoi fraktsii pecheni krysy.

Using isoelectrofocusing, the existence of multiple forms of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) in the postmitochondrial fraction of rat liver has been demonstrated for the first time. The isoelectric points for the enzyme isoforms are 6.25, 7.5 and 8.25.

[Does malonate take part in the synthesis of sterols?]. / Uchastvuet li malonat v sinteze sterinov?

The effect of acetyl-CoA-carboxylase activators, citrate and biotin, on cholesterol biosynthesis from acetate and malonate in rat liver and in cultured human lung fibroblasts was studied. Administration of citrate and biotin to animals and an addition of biotin to the fibroblast culture medium led to a significant stimulation of [2-14C]acetate incorporation into sterols but had no effect on the incorporation of [2-14C]malonate. The data obtained suggest that malonate is incorporated into sterols without preliminary decarboxylation.

[The role of different pathways of mevalonate synthesis in the regulation of sterol and bile acid synthesis in the mammalian liver]. / Znachenie razlichnykh putei sinteza mevalonata v reguliatsii obrazovaniia sterinov i zhelchnykh kislot v pecheni mlekopitaiushchikh.

The role of various pathways of synthesis are considered for mevalonic acid, the first specific precursor of sterols, in the production of cholesterol and bile acids in the mammalian liver. It is emphasized that the mevalonate synthesis with participation of acetyl-CoA-carboxylase and hydroxymethylglutaryl-CoA-reductase not bound with the endoplasmic reticulum membranes results in formation of the pool of mevalonic acid and other precursors necessary mainly for the organism supply with bile acids under conditions of cholesterol synthesis inhibition.

[Biosynthesis of mevalonic acid, sterols and bile acids from acetyl-CoA and malonyl-CoA in the human liver]. / Biosintez mevalonovoi kisloty, sterinov i zhelchnykh kislot iz atsetil-CoA i malonil-CoA v pecheni cheloveka.

The biosynthesis of mevalonic acid, squalene, sterols, bile and fatty acids from [2-14C]malonyl-CoA and [1-14C]acetyl-CoA were studied. The activities of 3-hydroxy-3-methylglutaryl-CoA-reductase (GMG-CoA reductase) and acetyl-CoA carboxylase in subcellular fractions of human liver were determined. The livers of humans were used within 1.5-3 hours after clinical death. It was found that in all fractions studied (i.e. cell-free, 700 g, postmitochondrial, microsomal, cytosol) malonyl-CoA is incorporated into mevalonic acid more intensively than acetyl-CoA. The specific activity of GMG-CoA reductase in the microsomal and soluble fractions was essentially the same. Calculation of enzymatic activity per 1 g of wet mass of tissue showed that the bulk of activity is bound to the cytosol (soluble fraction) Malonyl-CoA can also act as a precursor of squalene, lanosterol, cholesterol and bile acids. The rate of malonyl-CoA incorporation into these compounds is practically the same as that of [2-14C] mevalonate but significantly exceeds that of acetyl-CoA at equal molar ratios of both substrates. Incorporation of malonyl-CoA into cholesterol occurs much more intensively in human liver than in rat liver, the cholesterol radioactivity reaching 18% of the total unsaponified fraction. Malonyl-CoA is a better substrate than acetyl-CoA both for fatty acid and for mevalonate, sterol and bile acid synthesis.

[Biosynthesis of cholic and chenodeoxycholic acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in a reconstituted system from the rat liver]. / Biosintez kholevoi i khenodezoksikholevoi kislot iz (1-14C)atsetil-CoA i (2-14C)malonil-CoA v rekonstruirovannoi sisteme pecheni krysy.

The possibility of biosynthesis of cholic (I) and chenodeoxycholic (II) acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in a reconstituted system of rat liver and the incorporation of acetyl-CoA into these bile acids under conditions of acetyl-CoA carboxylase activation by citrate or its inhibition by avidin were studied. The effects of Triton WR 1339 and cholesterol feeding on acetyl-CoA and malonyl-CoA incorporation into I and II were investigated. Teh incorporation of both substrates into the total unsaponifiable lipid fraction and fatty acids was demonstrated. The reconstituted system of rat liver was found able to synthesize and I and II not only from acetyl-CoA, but from malonyl-CoA as well. The rate of malonyl-CoA incorporation into the bile acids was somewhat higher than that of acetyl-CoA incorporation. Preincubation of the reconstituted system with citrate stimulated the rate of acetyl-CoA incorporation into I. Stimulation of biosynthesis of I occurred independently of the diurnal rhythm of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) activity. An addition of avidin to the reconstituted system preincubated with citrate caused inhibition of acetyl-CoA incorporation both into fatty acids and into I. The rate of biosynthesis of II remained practically unchanged in both cases. Treatment with Triton WR 1339 had only a slight effect, while cholesterol feeding significantly stimulated the incorporation of acetyl-CoA and malonyl-CoA into I and II. The results obtained suggest the participation of malonyl-CoA in formation of bile acids, preferentially cholic acid, and in a lesser degree, in sterol biosynthesis. Data from stimulation of bile acid biosynthesis under cholesterol feeding suggest that HMG-CoA reductase localized in the soluble fraction of rat liver is involved in bile acid biosynthesis.

[Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet]. / Aktivnost' 3-gidroksi-3-metilglutaril-CoA-reduktazy, atsetil-CoA-karboksilazy i skorost' biosinteza mevalonovoi kisloty, skalena, sterinov i zhirnykh kislot iz [1-14C]atsetil-CoA i [2-14C]malonil-CoA v pecheni krysy: vliianie vvedeniia tritona WR 1339 golodaniia i kormleniia kholesterinom.

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.

[Activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and rate of biosynthesis of mevalonic acid, squalene, sterols and fatty acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: changes induced by daily rhythm]. / Aktivnost' 3-gidroksi-3-metilglutaril-CoA-reduktazy, atsetil-CoA-karboksilazy i skorost' biosinteza mevalonovoi kisloty, skvalena, sterinov i zhirnykh kislot iz [I-14C]atsetil-CoA i [2-14C]malonil-CoA v pecheni krysy: izmenenie v zavisimosti ot vremeni sutok.

The activity of 3-hydrosy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and the rate of mevalonic acid (MVA) synthesis from [I-14C]acetyl-CoA and [2-14C]malonyl-CoA in the soluble (X140000 g) and microsomal fractions of rat liver and in a reconstituted system containing the soluble and microsomal fractions were studied. The changes in the activity of HMG-CoA reductase and the rate of MVA biosynthesis in the fractions at different times of the day were analyzed. The daily rhythms of the rate of acetyl-CoA and malonyl-CoA incorporation into squalene, sterols and fatty acids in the postmitochondrial fraction and the daily changes in the acetyl-CoA carboxylase activity of the soluble fraction of rat liver were compared. The incorporation of labelled acetyl-CoA and malonyl-CoA into MVA showed that the latter can be synthesized from these two substrates both in the soluble and microsomal fractions. Malonyl-CoA is a preferable substrate for MVA synthesis in the soluble fraction. MVA synthesis from acetyl-CoA proceeds fastr in the intact and solubilized microsomes than in the soluble fraction. The activity of HMG-CoA reductase was found in the soluble and microsomal fractions in practically equal amounts. The enzyme activity was increased in the microsomal fraction after its solubilization. The rate of MVA biosynthesis from acetyl-CoA and the activity of HMG-CoA reductase in the soluble fraction are practically unaffected by day-to-night changes. The activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA in the intact and solubilized microsomal fractions reached their maximal values in the middle of the dark period. The rate of MVA biosynthesis from malonyl-CoA was decreased in the middle of the dark period in all fractions studied and reached its maximum in the middle of the light period. The daily rhythms of the acetyl-CoA carboxylase activity in the soluble fraction and the rate of MVA biosynthesis from malonyl-CoA in all fractions show a coincidence. a comparison of incorporation by the postmitochondrial fractions of acetyl-CoA and malonyl-CoA into the total non-saponified lipid fraction and its components, e. g. squalene, lanosterol and cholesterol, as well as into sterols precipitated by digitonin, showed that malonyl-CoA incorporation into the total non-saponified lipid fraction was more intensive than that of acetyl-CoA. However, acetyl-CoA was far more efficiently incorporated into sterols precipitated by digitonin or isolated by TLC than malonyl-CoA. The rate of acetyl-CoA incorporation into the total non-saponified lipid fraction and into squalene, lanosterol and cholesterol was maximal in the middle of the dark period and minimal in the middle of the light period. On the contrary, the rate of malonyl-CoA incorporation into these products was minimal in the middle of the dark period and maximal in the middle of the light period. The rate of fatty acid biosynthesis from acetyl-CoA was increased in the middle of the light and dark periods...

[Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]. / Dekarboksilirovanie malonil-koA i biosintez mevalonovoi kisloty v pecheni krys.

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.

[The activity of beta-hydroxy-beta-methylglutaryl-CoA reductase in the soluble fraction of rat liver]. / Aktivnost' beta-gidroksi-beta-metilglutaril-KoA-reduktazy v rastvorimoi fraktsii pecheni krysy

Assay conditions are worked out for determination of activity of beta-hydroxy-beta-methylglutaryl-CoA reductase (HMG-CoA reductase) in 140.000 g supernatant fraction of the rat liver. Some kinetic properties of the enzyme are studied: the activity dependency on the incubation time, protein concentration, pH, glutathione, dithiothreitol and HMG-CoA contents in the incubation medium. The effect of Triton WR 1339 on the activity of HMG-CoA reductase in the liver 140.000 g supernatant and microsomal fractions is comparatively studied. Diurnal activity variations of soluble and microsomal enzymes are also investigated. It is suggested that the rat liver HMG-CoA reductase in the 140.000 g supernatant fraction is not identical to the enzyme located in the microsomal fraction.

[Possible role of acetyl-CoA-carboxylase in biosynthesis of mevalonic acid and sterols in rat liver]. / O vozmozhnom uchastii atsetil-KoA-karboksilazy v biosinteze mevalonovoi kisloty i sterinov v pecheni krysy.

Effect of citrate on acetyl-CoA incorporation into mevalonic acid, sterols and fatty acids after preliminary incubation of rat liver extracts under conditions optimal for acetyl-CoA carboxylase activation, was studied. 30 min preincubation with the citrate at 37 degrees C results in a 2--3-fold stimulation of the mevalonic acid biosynthesis from acetyl-CoA in the microsomal and soluble (140 000 g) fraction, and in that of sterols precipitated by digitonin or isolated by TLC in the mitochondria--free fraction. 2-14C-malonyl-CoA incorporation into the mevalonic acid and sterols and biosynthesis of sterols from 2-14C-mevalonic acid were not stimulated under those conditions. A correlation was shown to exist between the activity of acetyl-CoA carboxylase and the rate of acetyl-CoA incorporation into mevalonate and sterols; the activity of beta-hydroxy-beta-methylglutaryl-CoA reductase, limiting the rate of the sterol biosynthesis, was not changed. The stimulating effect of citrate was found to depend on the concentration of acetyl-CoA and NADPH in the medium. The data obtained suggest that the mevalonic acid biosynthesis in rat liver may occur in the presence of acetyl-CoA carboxylase through the formation of malonyl-CoA.