George Popják

Cholesterol biosynthesis in human lymphocytes, monocytes, and granulocytes.

Abstract Lymphocytes, monocytes and granulocytes were separated by counter-flow centrifugation from the blood of normal individuals and were incubated in full serum medium or lipid-depleted medium. The monocytes incorporated about five times more [2- 14 C]acetate into sterols than did the lymphocytes in full serum medium and approximately twenty times more than the lymphocytes in lipid-depleted medium. The granulocytes were unable to synthesize sterols from either [2- 14 C]acetate or [2- 14 C]mevalonate, but they were able to use these substrates for the synthesis of squalene and demonstrated approximately a two fold increase in the incorporation of [2- 14 C]acetate (but not [2- 14 C]mevalonate) into squalene when incubated in the lipid-depleted medium as compared to the full serum medium.

Human liver prenyltransferase and its characterization

Prenyltransferase (dimethylallydiphosphate: isopentenyldiphosphate dimethylallytransferase, EC 2.5.1.1) has been purified to homogeneity from human liver obtained at autopsy. The enzyme is a dimer with a native molecular weight of 74 000 +/- 1 400. The amino acid composition is reported. the enzyme has a broad pH optimum between 7.3 and 8.8 and an absolute requirement for either Mn2+ or Mg2+ for activity; half-maximal activity was observed at 3.7 microM Mn2+ or 89.0 microM Mg2+. Michaelis constants for geranyl pyrophosphate and isopentenyl pyrophosphate were 0.44 and 0.94 microM, respectively; the V value for synthesis of farnesyl pyrophosphate from these substrates was 1.1 mumol . min-1 . mg-1. Isopentenyl pyrophosphate inhibited the reaction rates at concentrations above 2 microM when the concentrations of geranyl pyrophosphate were less than 2 microM. The highest concentration of geranyl pyrophosphate tested, 16 microM, showed no inhibition of reaction rates even when the concentration of isopentenyl pyrophosphate was as low as 0.2 microM. Only one form of human liver prenyltransferase could be observed under conditions which resolved the porcine enzyme into two distinct forms; the human enzyme is akin, physico-chemically, to the B-form of the pig liver enzyme. After dialysis against Tris-HCl buffer, pH 7.8, the enzyme became completely dependent upon dithiols or thiols for its activity. Kinetic experiments with a partially activated enzyme sample showed that the activation by the dithiol greatly enhanced the affinity of the enzyme for geranyl pyrophosphate, but not that for isopentenyl pyrophosphate. The human prenyltransferase is inactivated by phenylglyoxal according to pseudo-first-order kinetics, but is protected against the inactivation by 3,3-dimethylallyl and geranyl pyrophosphate. It is also inactivated by high concentrations (greater than 2 mM) of iodoacetic acid, but is protected against the inactivation by dithiothreitol. Antibodies raised to the B-form of the pig liver enzyme cross-reacted with the human prenyltransferase and were 47% as effective in precipitating the human enzyme as the porcine enzyme. In double immunodiffusion experiments the antiserum was monospecific against the B-form of the porcine enzyme; it also gave a single precipitin line with the A-form, but not identical with that given by the B-form. It gave a precipitin line also with the human enzyme, but not identical with that given by either the A- or B-form of the porcine enzyme.

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of Sendai virus as fusogen.

A method of introducing enzymes into the cytoplasm of fibroblasts in culture is described. Erythrocytes obtained from normal and arginase-deficient individuals were loaded with arginase in vitro and fused to arginase-deficient mouse and human fibroblasts. Erythrocyte ghost-fibroblast fusion was quantified by a 14C-radioactive assay for arginase in solubilized fibroblasts. Fusion was successfully induced by Sendai virus and also by the isolated glycoproteins of Sendai virus. After fusion the arginase activity associated with the Fibroblasts was 700--1500 U of arginase/mg of cell protein; this enzyme activity was 5- to 10-times higher than that normally found in the fibroblasts. The enrichment in arginase activity indicated that between four and ten ghosts had fused per fibroblast. The use of isolated viral proteins to mediate the transfer of enzymes into cells in vivo might alleviate clinical complications inherent in the use of whole virions. The enzyme replacement technique described in this report for a hyperargininemic model cell system should be applicable to the group of inborn errors of metabolism characterized by deficiency of an enzyme normally localized in the cytoplasmic compartment of cells.

A direct relationship between the amount of sterol lost from rat hepatocytes and the increase in activity of HMG-CoA reductase

Abstract Incubation of rat hepatocytes in a sterol-free medium containing 1.5% albumin resulted in loss of cholesterol from the cells and increased activity of HMG-CoA reductase. Addition of egg-lecithin dispersions to the hepatocytes resulted in increased rates of sterol efflux and increased levels of reductase. The increase in enzyme activity after three hours incubation was directly proportional to the amount of cholesterol lost by the cells to the medium during the first 45 min of incubation. Sterol loss preceded the increase in enzyme activity. The data support the view that one mode of regulation of hepatic HMG-CoA reductase is dependent on the relative rates of movement of cholesterol into and out of cells.

Pseudo-isoenzyme forms of liver prenyl transferase.

Abstract Prenyl transferase of pig liver exists in two forms, A and B, the latter being more acidic than form A. The ratio of these two forms in initial extracts of the organ varies according to the presence or absence of a thiol reducing agent in the extraction medium. In the presence of 10 mM β-mercaptoethanol the two forms exist as an equilibrium mixture of ca. 70% form A and 30% form B. The latter, when separated and rechromatographed with β-mercaptoethanol reverts to approximate the same 7:3 ratio of forms A and B. In the absence of a thiol reducing agent form B does not change, but form A may be converted almost completely to form B after treatment with oxidized glutathione.

Characterization of liver prenyl transferase and its inactivation by phenylglyoxal

The two interconvertible forms of pig liver prenyl transferase, A and B, consist of two identical subunits of Mr = 38 500 and are dimers. Form A contains six titratable SH-groups, whereas form B contains only four per dimer. The amino acid composition of the two forms is otherwise identical. Both enzyme forms are inactivated by phenylglyoxal. The inactivation in the absence of Mg2+ or Mn2+ is biphasic, each phase following pseudo first-order kinetics and is accompanied by a proportional binding of [14C]phenylglyoxal to the protein. In the initial fast phase of inactivation (t1/2 = 9.6 min) the amount of [14C]phenylglyoxal bound to the enzyme extrapolated to 1.1 arginyl residues and in the second phase (t1/2 = 23 min) to 2.2 arginyl residues modified per subunit for complete inactivation. 1 mM Mg2+ and 0.1 mM Mn2+ abolished the initial fast rate of inactivation and reduced its rate to a single half-life of about 60 min. Even at this slow rate of inactivation in the presence of Mg2+, the amount of [14C]phenylglyoxal bound to the enzyme extrapolated to about 2.3 arginyl residues modified per subunit for complete inactivation. In the absence of Mg2+ or Mn2+ only 1 mM geranyl pyrophosphate protected the enzyme against inactivation. However, in the presence of 1 mM Mg2+, isopentenyl, dimethylallyl and geranyl pyrophosphates gave additional protection over that observed with the metal ions, geranyl pyrophosphate being the most effective at 0.1 mM concentration.

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.

Non-sterol metabolism of mevalonate in vitro: Artifacts and realities☆

Abstract Commercial [5-14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated R -5-phospho-[5- 14 C]mevalonate by ion-exchange chromatography. The artifactual 14 CO 2 results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the 14 CO 2 and [ 14 C ]cholesterol produced by rat renal cortex slices incubated with purified [5-14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that R [5- 14 C]mevalonate is genuinely oxidized to 14 CO 2 in vitro , and that purification of substrate before its use is necessary. Production of 14 CO 2 and various [ 14 C ]lipids from purified [5-14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.

Human red cell galactose 1-phosphate uridylyltransferase: Effects of site-specific reagents on catalytic activity

Abstract Inactivation studies with the group-specific reagents p-chloromercuribenzoate, iodoacetic acid, and diethylpyrocarbonate (ethoxyformic anhydride) have been performed on purified human red cell galactose 1-phosphate Uridylyltransferase. The enzyme is rapidly inactivated by all three reagents at 17 °C. Complementary substrate protection studies using UDP-glucose, UDP-galactose, and galactose 1-phosphate show that none of these substrates exerts any detectable protective effect against inactivation by p-chloromercuribenzoate, but the UDP-hexose substrates specifically shield the enzyme against inactivation by iodoacetic acid and diethylpyrocarbonate. Whereas the protection afforded to the enzyme by treatment with 1 m m amounts of the UDP-hexose substrates is only partial against inactivation by diethylpyrocarbonate, the protection against alkylation by iodoacetic acid is dramatic and complete at a substrate concentration between 25 to 50 μ m . However, the extent of alkylation is dependent upon the presence of dithioerythritol and in the absence of this reducing agent, only about 30% inactivation is possible. Dithioerythritol is also necessary for maximal catalytic activity. It is concluded that the enzyme contains both cysteinyl and histidyl residues within the active center of the enzyme and that these residues are essential for catalytic activity. It is presumed that the requirement for dithioerythritol is to reduce a disulfide bridge at or near the UDP-hexose binding site and expose an essential cysteinyl residue which is required for productive catalysis.

Preferential uptake and utilization of mevalonolactone over mevalonate for sterol biosynthesis in isolated rat hepatocytes

Abstract Mevalonolactone at micromolar concentrations is taken up by rat hepatocytes and is converted into non-saponifiable lipids much faster than mevalonate. Although the first evidence of decarboxylation of both mevalonolactone and mevalonate (as determined by the release of 14CO2 from the 1-14C-labelled substrates) was observed at the same time (2 min), the subsequent rate of decarboxylation of mevalonolactone was approx. 43-fold of that found with mevalonate. The more rapid utilization of micromolar concentrations of mevalonolactone, compared to mevalonate, can be partly explained by the approx. 2-fold faster entry of the unchanged mevalonolactone into intact cells compared to the anionic mevalonate. This difference in uptake into the cell was observed with both the pure (R)- and the biologically inactive (S)-enantiomers1 and was independent of temperature. At these micromolar concentrations of the lactone the cell sterol biosynthetic pathway was not saturated and approx. 66% of the (R)-enantiomer was converted within 12 min to either isopentenyl pyrophosphate or non-saponifiable lipids. However, chemical (i.e. non-enzymic) hydrolysis of the lactone is slow, has a half-life of approx. 60 min at 37°C and at pH 7.4 and results in less than 35% of the lactone being converted to the anion mevalonate during a 15 min incubation. Hence the rapid conversion of approx. 66% of the mevalonolactone into sterols can be most easily explained if the cells contain a mevalonolactone hydrolase.