RESUMO
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.
Assuntos
Cisteína/análogos & derivados , Diterpenos/análise , Hidrazinas , Ácido Mevalônico/metabolismo , Prenilação de Proteína , Proteínas/química , Animais , Células CHO , Cricetinae , Cisteína/análise , Indicadores e Reagentes , Cinética , Espectrometria de Massas , Metilação , Proteínas/isolamento & purificação , Proteínas/metabolismoRESUMO
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.
Assuntos
Metabolismo dos Lipídeos , Lipoproteínas/biossíntese , Ácido Mevalônico/metabolismo , Prenilação de Proteína , Animais , Células CHO , Colesterol/metabolismo , Cricetinae , Cisteína/análogos & derivados , Cisteína/metabolismo , Diterpenos/metabolismo , Relação Dose-Resposta a Droga , Proteína Oncogênica p21(ras)/biossínteseRESUMO
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.