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G Popják

Publications and source records attributed to G Popják.

At least 19 recordsLinked to original sources
PubMed1991

In vitro identification of a soluble protein:geranylgeranyl transferase from rat tissues.

The gamma subunit of mammalian trimeric G proteins has been shown previously to be modified in vivo on a cysteine residue situated at the carboxyl-terminal sequence-Cys-Ala-Ile-Leu-COOH by a 20-carbon prenyl moiety geranylgeranyl (Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S., and Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5873-5877; Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K-K., Clarke, S., Gelb, M. H., and Glomset, J. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5866-5872). A biotinylated peptide acceptor comprising the eight carboxyl-terminal amino acids of the gamma subunit and tritiated geranylgeranyl diphosphate were utilized to monitor a protein:prenyl transferase activity in rat organs of varying age. The transferase activity was dependent upon the presence of divalent metal ions and maximal activity was achieved with either 1 mM ZnCl2 or 20 mM MgCl2. Activity was shown to be linear with respect to time, protein concentration, substrate concentration, and the pH optimum was 7.5. Protein:geranylgeranyl transferase activity was detected in all rat organs studied with the highest specific activity in brain S100. No activity was detected in the membrane fraction. The specific activity in brain, liver, kidney, and heart increased with age. Radioactivity incorporated into the peptide acceptor from both [1-3H]geranylgeranyl diphosphate and [5-3H]mevalonate by 21-day-old rat brain S100 was released by treatment with methyl iodide, and in both cases, analysis of the cleavage products by reversed phase high performance liquid chromatography showed a peak of radioactivity co-eluting with a geranylgeraniol standard which was well resolved from a farnesol standard. This indicated that the rat brain S100 contained not only the protein:geranylgeranyl transferase but also geranylgeranyl synthetase activity and that the peptide acceptor was specific for geranylgeranyl under the conditions tested.

Age Factors↗
PubMed1979

Squalene synthetase.

In the first part of the review the background to the discovery of the asymmetric synthesis of squalene from two molecules of farnesyl pyrophosphate and NADPH is described, then the stereochemistry of the overall reaction is summarized. The complexity of the biosynthesis of squalene by microsomal squalene synthetase demanded the existence of some intermediate(s) between farnesyl pyrophosphate and squalene. This demand was satisfied by the discovery of presqualene pyrophosphate, an optically active C30 substituted cyclopropylcarbinyl pyrophosphate, the absolute configuration of which at all three asymmetric centers of the cyclopropane ring was deduced to be R. Possible mechanisms for the biosynthesis of presqualene pyrophosphate and its reductive transformation into squalene are presented. In the second part of the review the nature of the enzyme is discussed. The question whether presqualene pyrophosphate is an obligate intermediate in the biosynthesis of squalene is examined, with the firm conclusion that it is. It is as yet uncertain whether the two half reactions of squalene synthesis, i.e. (i) 2 x farnesyl pyrophosphate leads to presqualene pyrophosphate; (ii) presqualene pyrophosphate + NADPH (NADH) leads to squalene, are catalyzed by one or two enzymes or by a large complex with two catalytic sites. Evidence is cited for the existence on the enzyme of two distinct binding sites with different affinities for the two farnesyl pyrophosphate molecules. The types of enzyme preparations available at present are described and types of experiments carried out with these are critically examined. The implications of the properties of a low molecular weight squalene synthetase solubilized with deoxycholate from microsomal membranes is discussed and a model for the enzyme in an organized membrane structure is presented.

Animals↗
PubMed1979

Determination of mevalonate in blood plasma in man and rat. Mevalonate "tolerance" tests in man.

A method is described for the determination of mevalonate in ultrafiltrates of blood plasma. The method depends on the phosphorylation of mevalonate with [gamma-32P]ATP and mevalonate kinase to 5-[32P]phosphomevalonate, and the subsequent isolation of the 5-[32P]phosphomevalonate together with known amounts of added 5-phospho[14C]mevalonate by ion-exchange chromatography. The 32P/14C ratio in the isolated 5-phosphomevalonate is a linear function of the mevalonate content of the samples. The smallest amount that can be determined is 1--2 pmol. The fasting level in human plasma varied between 20 and 75 pmol/ml. Human red blood cells absorb mevalonate from plasma relatively slowly; their maximum storage capacity is about 1.3 pmol/10(6) red cells. An oral and intravenous "mevalonate tolerance test" in man is described that can be carried out with 200 and 30 mumol. respectively, of the unlabeled (RS)-mevalonate in a 70-kg man. Beer and wine contain mevalonate at a concentration of 3--8 microns, too low to provide a significant amount of mevalonate even for heavy drinkers. The mevalonate content of the plasma from the blood of the vena cava inferior of male rats varied between 81 and 502 pmol/ml and is positively related to the levels of liver 3-hydroxy-3-methylgultaryl-CoA reductase, suggesting that the liver is probably the main source of mevalonate circulating in blood. The plasma of renal venous blood contained only 33--85% as much mevalonate as the arterial plasma.

Animals↗
PubMed1978

Inhibition of liver prenyltransferase by alkyl phosphonates and phosphonophosphates.

n-Pentyl and n-decyl phosphonate and the corresponding phosphonophosphates were found to inhibit cholesterol synthesis from mevalonate in the 10000 X g supernatants of liver homogenates and the synthesis of farnesyl pyrophosphate from geranyl and isopentenyl pyrophosphate by purified liver prenyltransferase. Kinetic analysis of the inhibition of prenyltransferase showed that the phosphonates and the phosphonophosphates interacted with two forms, or two sites, of the enzyme. The order of increasing potency was C5-phosphonate less than C10-phosphonate less than C5-phosphonophosphate less than C10-phosphonophosphate. The phosphonophosphates were at least ten times stronger inhibitors than the phosphonates.

Animals↗
PubMed1978

Squalene synthetase. Solubilization from yeast microsomes of a phospholipid-requiring enzyme.

Squalene synthetase was solubilized from yeast microsomal membranes with deoxycholate. Solubilized enzyme was associated with one or more proteins with s20, w = 3.3 S, Stokes' radius = 40 A, and computed molecular weight = 54,500. In the presence of detergent the enzyme was catalytically inactive and unstable to heat. When detergent was removed with cholestyramine resin, both phases of squalene synthesis (farnesyl pyrophosphate leads to presqualene pyrophosphate leads to squalene) were recovered, and the enzyme was reaggregated to form sedimentable particles with a density of approximately 1.16 g/ml. Both activities were lost to variable extent upon chromatography over Sephadex G-200 in the presence of 0.2% deoxycholate, but could be recovered if phosphatidylcholine or phosphatidylethanolamine (but not phosphatidylserine or phosphatidylinositol) were added to fractions before removal of detergent. There was an apparently absolute requirement for phospholipid by the enzyme. The proteins catalyzing the two phases of squalene synthesis could not be resolved from one another and behaved in an identical fashion throughout a variety of manipulations.

Chemical Phenomena↗
PubMed1977

Control of 3-hydroxy-3-methylglutaryl coenzyme A reductase by endogenously synthesized sterols in vitro and in vivo.

Isolated rat hepatocytes converted mevalonolactone into sterol intermediates and fatty acids 6- to 8-fold faster than mevalonate salt at concentrations less than 6 X 10(-4) M. Incubation of hepatocytes for 3 h normally results in induction of 3-hydroxy-3-methylglutaryl-CoA reductase. This increase in enzyme activity was inhibited by mevalonolactone and by mevalonate salt; at each concentration between 6 X 10(-4) M and 6 X 10(-8) M the lactone was a more effective inhibitor than the salt. The increase in enzyme activity was completely prevented by 6 X 10(-4) M lactone, and at this concentration the cells synthesized from the lactone an amount of sterol per hour which approximated that leavingthe cells in the same period. Administration of mevalonolactone to intact rats resulted in a dose-dependent inhibition of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity. At the highest dose (400 mg of (RS)-mevalonolactone/200 g of rat) enzyme activities declined 85% within 45 min and were still suppressed below normals after 28 h. Mevalonolactone treatment resulted in increases in liver cholesterol content and in the cholesterol ester concentration of liver microsomes. The results demonstrate that the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase can be controlled by the rate of endogenous sterol synthesis both in vitro and in vivo.

Alcohol Oxidoreductases↗
PubMed1977

Mechanism of induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human leukocytes.

Incubation of leukocytes in buffer alone devoid of lipoproteins does not lead to the induction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, but incubation of these cells in lipid-depleted serum, abetaliprproteinemic serum, or lipoprotein-deficient serum (d greater than 1.21) leads to sterol loss from the cells and the activation of sterol synthesis from acetate. The latter was shown previously to be proportional to the HMG-CoA reductase levels in the cells (Fogelman, A. M., Edmond, J., Seager, J., and Popják, G. (1975)J. Biol. Chem. 250, 2045-2055). Sterol loss occurs from normal and heterozygous familial hypercholesterolemic leukocytes within 15 min in lipid-depleted serum. Since induction of HMG-CoA reductase activity is not detectable until after the leukocytes have been incubated in the lipid-depleted serum for at least 3 h (Fogelman et al., see above), sterol loss clearly precedes the induction of the enzyme. In six out of six experiments, the nonisotopic sterol content of leukocytes incubated in lipid-depleted serum was equal to or lower than that of the same leukocytes incubated in full serum. This occurred at a time when the leukocytes in the lipid-depleted serum were incorporating 4 to 5 times more [14C]acetate into sterols than the same leukocytes in full serum. This strongly suggests that the induction of the reductase was a compensatory mechanism for sterol loss. Incubation of leukocytes in buffer, or buffer plus lecithin dispersions, or buffer plus albumin did not lead to sterol loss or induction of the reductase, but incubation in buffer and albumin together with lecithin dispersions caused sterol loss into the medium and the activation of sterol synthesis from acetate. It is concluded that a phospholipid-protein-cell interaction, which produces sterol loss, is necessary to induce the reductase in leukocytes. A close correlation between sterol loss and total sterol synthesis (a function of HMG-CoA reductase activity) was demonstrated in normal and heterozygous leukocytes incubated in a variety of incubation media (r = 0.95; p less than 0.005). Heterozygous leukocytes taken fresh from the blood contained no more cholesterol than the leukocytes of their age and sex-matched controls, despite the marked difference in their serum cholesterol concentrations. It is proposed that the abnormality in familial hypercholesterolemia can be accounted for by an abnormal efflux of cholesterol from heterozygous cells.

Adolescent↗
PubMed1976

Properties of phenylalanine hydroxylase of cultured hepatoma cells.

The kinetic and immunologic properties of phenylalanine hydroxylase of adult rat liver were compared to the properties of the similar enzyme present in cultured H4-II-E-C3 hepatoma cells. The enzymes from the two sources could not be distinguished by the Km values for either phenylalanine or 6,7-dimethyltetrahydropterin. Analysis by double immunodiffusion showed that phenylalanine hydroxylase from the two sources had identical immunologic determinants, but immunotitrations revealed a small but significant difference between the enzyme of the normal adult rat liver and the enzyme of cultured hepatoma cells. The results of double immunodiffusion and immunotitration experiments indicated also that the increased levels of phenylalanine hydroxylase seen in the hepatoma cells grown in the presence of hydrocortisone resulted from the accumulation of enzyme protein, but it could not be decided whether this accumulation resulted from an increased rate of synthesis or decreased rate of degradation.

Animals↗
PubMed1976

Mevalonate metabolism: role of kidneys.

More than one-half of the amount of mevalonate that is metabolized by pathways not leading to sterols is accounted for by the action of the kidneys. Conversion of mevalonate in vivo to squalene and sterols in the kidneys is confined almost entirely to the proximal and distal convoluted tubules in the cortex. More sterol than squalene is synthesized from mevalonate not only in the liver but also in the kidney.

Animals↗