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.

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.

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.

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.

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.