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.

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells.

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.

Concerning the role of 24,25-dihydrolanosterol and lanostanol in sterol biosynthesis by cultured cells.

Rat hepatoma cells (H4-II-E-C3) efficiently converted a dietary supplement of [2-3H]24,25-dihydrolanosterol (1) to [3H]cholesterol while [2-3H]lanostanol (4,4,14 alpha-trimethylcholestanol (2) was recovered from the cells without apparent transformation, although it was esterified and induced an accumulation of lanosterol. A comparison of the chromatographic (TLC, GLC and HPLC), spectral (MS and 1H-NMR) and physical properties of 1 and 2 is given for the first time. The inability to detect 2 in nature coupled with our findings that 1 but not 2 is metabolized to cholesterol by H4 cells is interpreted to imply that the biosynthetic inclusion of the delta 8(9)-bond during the cyclization process of squalene-oxide to a tetracyclic product is an evolutionary adaptation selected for because the olefinic linkage is structually important in the subsequent conversion of lanosterol and its stereoisomers, e.g., cycloartenol, to delta 5-sterols.

Effects of 2,3-iminosqualene on cultured cells.

2,3-Iminosqualene (ISq) is a powerful inhibitor of squalene oxide:lanosterol cyclase (EC 5.4.99.7). When added to lipid-depleted culture media (LDM) of rat hepatoma (H-4-II-E-C3) or Chinese hamster ovary (CHO) cells at a concentration of 10 micrograms ml-1, it causes the cells to float off the substratum in a few days. Lipoproteins in the culture medium completely counteract this effect. Cells in lipoprotein-containing media (FGM) grow normally in the presence of ISq. Irrespective of the culture medium, ISq at 10 micrograms ml-1 causes an almost complete and apparently irreversible inactivation of the squalene oxide cyclase in CHO and H4 cells and the accumulation in the cells of squalene, of squalene 2,3-oxide (mostly), and of squalene 2,3-22,23-dioxide when [14C]acetate or [14C]mevalonate is fed to the cells. Chronic treatment of H4 cells with ISq failed to elicit induction of the cyclase, but increased the conversion of mevalonate into squalene and squalene dioxide, and depressed the conversion of squalene oxide to the dioxide. Cells loaded with squalene and the squalene oxides from mevalonate in the presence of ISq get rid of these substances by rapidly secreting them into the media and by some unidentified metabolic processes.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: search for the enzyme's repressor derived from mevalonate.

Three inhibitors of squalene 2,3-oxide-lanosterol cyclase (AMO 1618, 4,4,10 beta-trimethyl-trans-decal-3 beta-ol (TMD) and 2,3-iminosqualene (ISq] were used to study effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and on sterol and polyprenyl synthesis from [14C]acetate and [14C]mevalonate in cultured rat hepatoma (H4) cells. After a 4 h exposure of cultures to AMO 1618 or TMD, followed by removal of the inhibitors, the utilization of [14C]acetate for synthesis of digitonin-precipitable sterols increased about twofold, an increase parallelled by the rise in HMG-CoA reductase. Mevalonate at 2.3 mM counteracted the effects of these inhibitors on the reductase. When (R)-[2-14C]mevalonate at 2.3 mM was included with the two inhibitors in the culture media, the cells were still able to synthesize cholesterol although in lesser amounts than the controls. In the presence of TMD the H4 cells also accumulated [14C]squalene 2,3-oxide and [14C]squalene 2,3-22,23-dioxide. ISq added to cells kept in full-growth medium (10 micrograms ml-1) caused an almost complete and irreversible inactivation of the squalene oxide-lanosterol cyclase but did not inhibit polyprenyl synthesis, as the amount of [14C]mevalonate converted into squalene, squalene 2,3-oxide, squalene 2,3-22,23-dioxide plus a little cholesterol was equal to the amount converted by control cells into cholesterol plus squalene. After a 24 h exposure of cells kept in full-growth medium to ISq (10 micrograms ml-1), the levels of HMG-CoA reductase rose about twofold. ISq completely abolished the suppressive effect of 2.3 mM (R)-mevalonate on the reductase. Chromatin isolated from cell nuclei contains cholesterol, which is renewed biosynthetically. It is argued that the suppressor of HMG-CoA reductase, derived from mevalonate, is a sterol and not a non-steroidal product of mevalonate metabolism.

Inhibition of liver prenyltransferase by citronellyl and geranyl phosphonate and phosphonylphosphate.

Citronellyl- and geranylphosphonic acids and the corresponding phosphonylphosphates were made and tested as inhibitors of liver prenyltransferase. Kinetic analysis showed that citronellyl- and geranylphosphonylphosphate were powerful inhibitors of the enzyme, and that they were competitive inhibitors with geranyl diphosphate and noncompetitive inhibitors with isopentenyl diphosphate. Two inhibition constants, representing the equilibria [E][I]/[EI] = K5 and [ES1][I]/[ES1I] = K9, have been defined for the inhibitors. For citronellylphosphonylphosphate, the value of K5 was 1.25 microM and K9 was 3.30 microM; for geranylphosphonylphosphate, K5 = 1.50 microM and K9 = 1.60 microM. The phosphonates were very poor linear mixed noncompetitive inhibitors with respect to both substrates of the transferase.

Role of mevalonate in regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured cells and their cytoplasts.

H4-II-E-C3 hepatoma cells in culture respond to lipid-depleted media and to mevinolin with increased sterol synthesis from [14C]acetate and rise of 3-hydroxy-3-methylglutaryl coenzyme A reductase levels. Mevalonate at 4 mM concentration represses sterol synthesis and the reductase, and completely abolishes the effects of mevinolin. Mevalonate has little or no effect on sterol synthesis or reductase in enucleated hepatoma cells (cytoplasts) or on reductase in cytoplasts of cultured Chinese hamster ovary (CHO) cells. The sterol-synthesizing system of hepatoma cell cytoplasts and the reductase in the cytoplasts of CHO cells were completely stable for at least 4 hr. While reductase levels and sterol synthesis from acetate followed parallel courses, the effects on sterol synthesis--both increases and decreases--exceeded those on reductase. In vitro translation of hepatoma cell poly(A)+RNAs under various culture conditions gave an immunoprecipitable polypeptide with a mass of 97,000 daltons. The poly(A)+RNA from cells exposed for 24 hr to lipid-depleted media plus mevinolin (1 microgram/ml) contained 2.8 to 3.6 times more reductase-specific mRNA than that of cells kept in full-growth medium, or cells exposed to lipid-depleted media plus mevinolin plus mevalonate. Northern blot hybridization of H4 cell poly(A)+RNAs with [32P]cDNA to the reductase of CHO cells gave two 32P-labeled bands of 4.6 and 4.2 K-bases of relative intensities 1.0, 0.61-1.1, 2.56, and 1.79 from cells kept, respectively, in full-growth medium, lipid-depleted medium plus mevinolin plus mevalonate, lipid-depleted medium plus mevinolin, and lipid-depleted medium. These values approximate the reductase levels of these cells. We conclude that mevalonate suppresses cholesterol biosynthesis in part by being a source of a product that decreases the level of reductase-specific mRNA.

Characterization of a glycoprotein fusogen isolated from Sendai virus.

After isolation from Sendai virus, the glycoproteins HN and F retained their ability to induce hemagglutination and both heterologous and homologous cell-cell fusion. Both methods for demonstrating cell fusion indicated that the isolated HN and F glycoproteins compared favorably with whole Sendai virus as a fusogen. Conditions affecting the degree of fusion were examined and optimized. Whole virus and isolated glycoprotein preparations were characterized by electron microscopy and by SDS-polyacrylamide gel electrophoresis. Lipid analysis of the glycoprotein preparations by thin layer chromatography and gas chromatography/mass spectrometry indicated that they were partially lipid-depleted during the isolation protocol and the ratio of cholesterol to phospholipid was higher than in the whole virus. A complete fatty acid analysis was performed on lipid extracts from whole virus and from glycoprotein preparations. Detergent was removed from the glycoproteins by dialysis and by incubation with Amberlite XAD-2 resin. The detergent content of the glycoprotein preparations was monitored by gas chromatography and with [3H]Triton X-100. Both methods showed that virtually all (greater than or equal to 99.8%) of the originally added detergent was removed. Electron microscopy of the negatively-stained HN and F preparations showed primarily spherical particles 120 +/- 20 A in diameter (range 80-250 A). Since no organization reminiscent of envelopes could be demonstrated, we conclude that the fusogenic activity of Sendai virus resides in the glycoproteins per se rather than in bilayer integrated lipid-protein complexes.

Isolation and identification of 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3. New metabolites of vitamin D3 produced by a C-24 oxidation pathway of metabolism for 1,25-dihydroxyvitamin D3 present in intestine and kidney.

Two new vitamin D metabolites were isolated in pure form from separate incubations of homogenates of chick small intestinal mucosa or rat kidney employing either 1 alpha,25-dihydroxyvitamin D3 (28 microM) or 1 alpha,24R,25-trihydroxyvitamin D3 as substrate (0.17-1.3 microM). The newly characterized compounds and the amounts isolated in pure form from separate isolations are, respectively: 1 alpha,25-dihydroxy-24-oxo-vitamin D3 (1,25(OH)2-24-oxo-D3), 147 micrograms from kidney and 4.2 and 40 micrograms from intestine; 1 alpha,23,25-trihydroxy-24-oxo-vitamin D3 (1,23,25(OH)3-24-oxo-D3), 155 micrograms from kidney and 5.9 and 34 micrograms from intestine. Their structures were identified after extensive high pressure liquid chromatography by means of ultraviolet absorption spectrometry, mass spectrometry of the free compounds and their trimethylsilylated derivatives, proton nuclear magnetic resonance spectrometry, specific chemical reduction of the 24-oxo functionality with sodium borohydride, as well as direct comparison with synthetic 1,25(OH)2-24-oxo-D3. These structural assignments for both compounds correct previous determinations which had been proposed (Ohnuma, N., Kruse, J. R., Popjak, G., and Norman, A. W. (1982) J. Biol. Chem. 257, 5097-5102). The activity of the C-24 oxidation pathway used for the production of the 1,25(OH)2-24-oxo-D3 and 1,23,25(OH)3-24-oxo-D3 can be enhanced 10-fold by prior priming of the chicks or rats with a single intravenous dose of 1,25(OH)2D3 (1-12 nmol/100 g body weight); the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels within 12 h. Further, 1,25(OH)2D3, 1,24,25(OH)3D3, and 1,25(OH)2-24-oxo-D3 all were found to be capable of serving as a precursor with chick intestine and rat kidney homogenates of 1,23,25(OH)3-24-oxo-D3. Collectively these results suggest the existence of a C-24 oxidation pathway for metabolism of 1,25(OH)2D3 by the target intestinal mucosa and kidney to 1,23,25(OH)3-24-oxo-D3. The pathway may play an important role in controlling the tissue levels of this hormonally active form of vitamin D3.

Transferase-deficiency galactosemia: evidence for the lack of a transferase protein in galactosemic red cells.

Red blood cell lysates from normal individuals, a homozygous Duarte variant, and a patient with transferase-deficiency galactosemia were challenged with rabbit antibody to pure human placental galactose-1-phosphate uridylyltransferase. Although the antibody quantitatively precipitated the enzymatically active proteins in the normal and Duarte hemolysates, the Duarte sample absorbed only about one-half as much antibody as did the normal. In contrast, the antibody did not react with the galactosemic hemolysate.

23,25-Dihydroxy-24-oxovitamin D3: a metabolite of vitamin D3 made in the kidney.

Kidney homogenates of rats produced a new metabolite of 25-hydroxyvitamin D3 which has been isolated in pure form after five column chromatographic steps. It was identified as 23,25-dihydroxy-24-oxovitamin D3 by means of ultraviolet and infrared absorption spectrophotometry, mass spectrometry, and proton nuclear magnetic resonance spectrometry. The stereochemistry at the C-23 position is as yet unknown. 25-Hydroxy-24-oxovitamin D3, which also has been isolated in pure form from this system, was found to be the precursor of the new metabolite in vitro. The production of the new metabolite was induced by two different methods: (a) perfusion of the kidneys with 1,25-dihydroxyvitamin D3 contained in the perfusate and (b) injection of 1,25-dihydroxyvitamin D3 in the intact animal. 23,25-Dihydroxy-24-oxovitamin D3 was not biologically active in an assay for intestinal calcium transport and bone calcium mobilization in the vitamin D deficient chick at a dose level of 5.3 nmol. A metabolic pathway is proposed to describe the results; it leads from 25-hydroxyvitamin D3 leads to 24(R),25-dihydroxyvitamin D3 leads to 25-hydroxy-24-oxovitamin D3 leads to 23,25-dihydroxy-24-oxovitamin D3.

Isolation and chemical characterization of two new vitamin D metabolites produced by the intestine. 1,25-Dihydroxy-23-oxo-vitamin D3 and 1,25,26-trihydroxy-23-oxo-vitamin D3.

Two new vitamin D metabolites were isolated in pure form from incubations of 53 nM 1,25-dihydroxyvitamin D3 with homogenates of small intestinal mucosa of vitamin D-replete chicks. The birds were injected intravenously with 8 to 9 nmol of 1,25-dihydroxyvitamin D3/100 g body weight 5 to 8 h before death. The isolation involved methanol-chloroform extraction and four successive chromatographic procedures (Sephadex LH-20 and high performance liquid chromatography). Chemical structures of the metabolites are proposed on the basis of (a) their chromatographic behavior, (b) their mass spectra, and (c) ultraviolet absorption spectra. They are identified as 1 alpha,25-dihydroxy-23-oxo-vitamin D3 and 1 alpha,25,26-trihydroxy-23-oxo-vitamin D3. Neither of the two new metabolites is produced by the intestinal mucosa when 1,25S,26-trihydroxyvitamin D3 is used as a substrate.

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.

Translation of phenylalanine hydroxylase-specific mRNA in vitro: evidence for pretranslational control by glucocorticoids.

We have found that the induction of phenylalanine hydroxylase by hydrocortisone and serum in confluent cultures of H4-II-E-C3 rat hepatoma cells is accompanied by an increase in polysomal mRNA specific for phenylalanine hydroxylase, as measured by translation in a cell-free protein-synthesizing system. Thus, the induction is mediated largely, if not entirely, by a pretranslational mechanism, possibly by stimulation of the transcription of the phenylalanine hydroxylase gene.

Characterization of peptides in human cerebrospinal fluid.

The report of endorphin-like activity in human CSF [7] has stimulated us to study peptides in various CSF specimens. At first we attempted to fractionate CSF by gel-filtration and paper-mapping procedures. By these techniques, we have isolated a peptide (Gly-Ala3-Val-Leu) contaminated with glutamic acid (or glutamine), which could not be removed by either of these methods. An HPLC method was developed for peptide fractionations on a RP-18 column with a gradient of 18 mM ammonium acetate and acetonitrile. This system has been evaluated with synthetic peptides of molecular weight up to about 1850, and has the advantages that the buffer salts may be removed by lyophilization and separations are performed at or near neutral pH thus minimizing alterations in the structure of the peptides. The retention characteristics of peptides on a C18 column is a function of the pH of the buffer. This facilitates the characterization of peptides since each may be identified by its unique chromatographic behavior at different pH values on one column.