Biomedical subjects

H C Rilling

Publications and source records attributed to H C Rilling.

At least 19 recordsLinked to original sources

PubMed1994

Thioprenols as hydrazinolysis products of prenylated proteins: dependence upon methylation of the prenylcysteine.

When prenylated proteins are treated with hydrazine at elevated temperatures, a substantial fraction of the prenylcysteines are cleaved at the C-S bond of the beta-carbon of cysteine. Thioprenols, the initial products of this reaction, are then reduced, over time, to hydrocarbons. This elimination reaction is favored several fold if the prenylcysteine is present as a carboxylate derivative rather than as a carboxyl terminal free amino acid. Thus, the pattern of elimination has the potential for detecting substitution (methylation) of prenylcysteines. In addition, the formation of thioprenols leads to more sensitive ways for determination of the prenylcysteines.

Animals↗

PubMed1993

Differential prenylation of proteins as a function of mevalonate concentration in CHO cells.

The incorporation of [5-3H]mevalonate into prenylated proteins and polyisoprenoid lipids has been determined as a function of mevalonate concentration in Chinese hamster ovary (CHO) cells that are inhibited in mevalonate synthesis. The relative incorporation of mevalonate into the different end products of isoprenoid metabolism was markedly dependent upon the concentration of mevalonate in the medium. The synthesis of cholesterol was dominant at higher concentrations of mevalonate while higher molecular weight isoprenoids were favored at the lower concentrations. The relative incorporation of mevalonate into the different prenylcysteines of prenylated proteins was dependent upon mevalonate concentration with geranylgeranylcysteine being the principal product at higher concentrations. At low levels of mevalonate farnesylcysteine synthesis predominated and geranylcysteine was detected. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from CHO cells that had been radiolabeled at different concentrations of [3H]mevalonate had different patterns on fluorography with relatively few proteins labeled at low concentrations. A study of this effect on the prenylcysteines of a specific protein, Ras, showed considerably less sensitivity to mevalonate concentration than bulk protein. These results indicate that the specific proteins that are prenylated depend upon the availability of the isoprenyl diphosphate substrates.

Animals↗

PubMed1991

Quantitation of prenylcysteines by a selective cleavage reaction.

The allylic thioether bond of the prenylcysteines of prenylated proteins has been shown to be cleaved by 2-naphthol under alkaline conditions to yield substituted naphthopyrans. These products are readily resolved from interfering materials by HPLC and have a strongly absorbing chromophore. Thus, this reaction is suitable for quantitative analysis of prenyl substituents of proteins, and we have examined a number of tissues for their content of prenylcysteines. These amino acids are present in mammalian tissues at a concentration of 0.36-1.4 nmol/mg of protein, with a ratio of geranylgeranylcysteine to farnesylcysteine in the range of 4 to 10. Prenylcysteines were also found in the cytosolic fraction of two mouse tissues at about one-third the concentration of the whole organ. The level of these modified amino acids was found to be significantly less in a yeast, a fungus, a brown alga, a higher plant, and an insect. Again, geranylgeranylcysteine is predominant. Prenylcysteines were absent from Escherichia coli but present in an archaebacterium. The prenylcysteine content of mammalian tissue is about 1% of that of cholesterol and about equal to that of ubiquinones and dolichols. Calculations indicate that about 0.5% of all proteins are prenylated.

3T3 Cells↗

PubMed1990

Prenylated proteins: the structure of the isoprenoid group.

The mevalonate-derived portion of a prenylated protein from Chinese hamster ovary cells has been established as diterpenoid (C20). This group is linked to a carboxyl-terminal cysteine as a thioether. It was removed from the protein by hydrazinolysis followed by Raney nickel desulfurization, and the resulting hydrocarbon fraction was analyzed by gas chromatography-mass spectrometry.

Animals↗

PubMed1990

Prenylated proteins: synthesis of geranylgeranylcysteine and identification of this thioether amino acid as a component of proteins in CHO cells.

Prenylated proteins, labeled in the isoprenoid residue by growing CHO cells in medium containing [5-3H]mevalonate, were degraded by three different proteolytic procedures, enzymatic or alkaline hydrolysis as well as hydrazinolysis. The products thus obtained were analyzed by HPLC with chemically prepared all-trans-geranylgeranylcysteine as a standard. About 10% of the radioactive products released by each lytic procedure showed the same chromatographic properties as geranylgeranylcysteine. This verifies the earlier conclusion, based on less-direct evidence, that this thioether derivative of cysteine is a component of naturally occurring proteins. The finding of this modified amino acid as a product of hydrazinolysis indicates that it is a carboxyl-terminal amino acid and that it is not carboxyl-methylated.

Animals↗

PubMed1989

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins.

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.

Animals↗

PubMed1988

Purification of geranylgeranyl diphosphate synthase from Phycomyces blakesleanus.

Geranylgeranyl diphosphate synthase has been purified to homogeneity from the carotene-overproducing strain M1 of Phycomyces blakesleanus. Usually two activity peaks with molecular weights of 60,000 and 30,000 eluted on gel exclusion chromatography, suggesting that the enzyme consists of two subunits, with a tendency to dissociate. With homogeneous protein, a single-staining band with molecular weight of 30,000 appeared on sodium dodecyl sulfate gel electrophoresis, confirming a subunit molecular weight of 30,000. Only isopentenyl diphosphate and farnesyl diphosphate were accepted by this enzyme for geranylgeranyl diphosphate formation. The smaller allylic compounds, dimethylallyl and geranyl diphosphate, were utilized at less than 1/20th the rate of farnesyl diphosphate. Michaelis constants of 9 microM for isopentenyl diphosphate and 60 microM for farnesyl diphosphate were found. The isoelectric point is 4.8.

Dimethylallyltranstransferase↗

PubMed1988

Purification to homogeneity and some properties of squalene synthetase.

Squalene synthetase has been purified to homogeneity from yeast. It is a single polypeptide of Mr 47,000. This enzyme catalyzes the synthesis of squalene from farnesyl diphosphate via presqualene diphosphate. In the presence of reduced pyridine nucleotides, presqualene diphosphate and squalene are produced in a ratio of 6:1 from either the purified protein or the crude microsomal fraction.

Chromatography↗

PubMed1988

Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis.

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 mumol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

Animals↗

PubMed1987

Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities.

Squalene synthetase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) is an intrinsic microsomal protein that catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate. We have solubilized this enzyme from yeast with a mixture of the detergents N-octyl beta-D-glucopyranoside and Lubrol PX. Approximately 50-fold purification of the solubilized activities has been achieved by chromatography on DEAE-cellulose and hydroxylapatite and by isoelectric focusing. The most highly purified preparation has one major band of protein with a molecular weight of 53,000 as estimated by electrophoresis under denaturing conditions. The enzyme may also have been modified by proteolysis during isolation since a 47,000 molecular weight species was also found. The two activities, presqualene pyrophosphate synthetase and squalene synthetase, copurified during isolation.

Chromatography, DEAE-Cellulose↗

PubMed1986

Prenylated proteins from kidney.

When [5-3H]mevalonate is injected into mice, it is incorporated into macromolecules in the kidney. The incorporated material is stable to treatment with RNase or DNase but not protease, indicating that the radioactivity is associated with protein. Electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels indicates a molecular weight of about 25,000. The incorporated radioactivity can be released from the polypeptide and extracted into organic solvents after hydrolysis with acid or base or by treatment with protease. The conditions required for hydrolysis strongly suggest that the linkage between the protein and the mevalonate-derived material is an allylic ether. The chromatographic mobility of the incorporated material in several systems is similar to that of dolichol C95.

Animals↗

PubMed1986

Isopentenyl pyrophosphate isomerase:dimethylallyl pyrophosphate isomerase: isolation from Claviceps sp. SD 58 and comparison to the mammalian enzyme.

Isopentenyl pyrophosphate isomerase:dimethyl pyrophosphate isomerase (EC 5.3.3.2) has been purified to near homogeneity from Claviceps sp. A molecular weight of 35,000 was found by gel exclusion chromatography as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the enzyme consists of a single subunit and is in contrast to the Mr 22,000 that we have found for the enzyme from liver. The lability of isomerase from liver, often reported, has been found to be due to its susceptibility to proteolysis. Nine compounds have been tested as inhibitors of both isomerases. The binding of analogs requires the pyrophosphate moiety which may be substituted by a variety of alkyl groups. Inclusion of a polar function in the hydrocarbon portion of the analog greatly reduces interaction with the enzyme. Reversibility of the reaction was not found with a higher homolog of the substrate.

Animals↗

PubMed1986

Prenyltransferase and squalene synthetase in livers of neonate rats.

The liver of the newly born rat has approximately the same capacity for cholesterol biosynthesis as that of the adult animal. However, during nursing, the ability to synthesize cholesterol diminishes markedly during the early neonate period and by the end of the second week has essentially vanished. The level of the regulatory enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA reductase, closely follows this pattern (Hahn, P. and Walker, B. (1979) Can. J. Biochem. 57, 1216-1219). In contrast, we have found that two other enzymes of cholesterol biosynthesis, prenyltransferase and squalene synthetase, undergo changes in activity that provide three maxima - one on birth, one during midnursing, and one on weaning. Possible explanations for this pattern are presented.

Acetates↗

PubMed1985