The aflylic 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 sigicantly less in a yeast, a fungus, a brown alga, a higher plant, and an insect. Again, geranylgeranylcysteine is predominant. Prenylcysteines were absent from Eschenchia coil but present in an archaebacterium. The prenylcysteine content of mmalan 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.Prenylated proteins contain isoprenoid-modified cysteines in the carboxyl-terminal region (1-3). This posttranslational modification has usually been identified by metabolic labeling of proteins of cells in culture with radioisotopic mevalonate. Metabolic labeling is limiting because many organisms-e.g., yeasts and plants-do not incorporate this precursor readily. In addition, this technique may be impractical, since large doses of isotope would be required for studies with animals. The specific isoprenoid involved with this amino acid (4, 5) has been established by protein hydrolysis and isolation of the prenylcysteine (6) or by cleavage of the prenylcysteine with iodomethane (7) or Raney nickel (4, 5) followed by isolation of the cleavage product. Recently, fast atom bombardment mass spectrometry has been reported as a convenient method for qualitative analysis of prenylated peptides (8). Quantitative analysis with these techniques by direct determination of mass would be difficult if not impossible. Consequently, an alternative method for identifying prenyl modifications would be useful. In prenylated proteins the allylic thioether bond of the prenylcysteines is unique and provides an avenue for selective reactions for identification of prenyl groups. A good nucleophile should react preferentially with and cleave the isoprenoid from cysteine. Prenyl groups linked through oxygen or nitrogen would be expected to be unreactive. For example, a model study using radioactive farnesyl pyrophosphate and our naphthoxide conditions showed about 0.1% of a radioactive product. If the reagent possessed a strongly absorbing chromophoric group, then the derivative would provide a means for detection of the product. We have allowed prenylated proteins to react with 2-naphthol and hav...

No abstract

A method has been developed to prepare short DNA sequences using light to deprotect a nucleoside 3‘-phosphotriester, generating a phosphodiester useful for coupling with a free 5‘-OH-nucleotide. The dimethoxybenzoin group is used as the photochemically-removable protecting group for the 3‘-phosphate. Cyanoethyl is most effective as the second protecting group on the phosphodiester. Because the method is directed at the preparation and use of the DNA sequences while still bound to the support, allyl and allyloxycarbonyl protecting groups are used for the nitrogenous bases since, based on the work of Hayakawa and Noyori, they can be removed without cleaving the DNA from the support. Two simple trinucleotides have been prepared in solution using this method. It has been demonstrated that the photochemical deprotection conditions do not lead to the formation of cyclobutane dimers from adjacent T residues.

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 carboxylterminal amino acid and that it is not carboxyl-methylated.

Posttranslational modification by prenylation is important in the activation of a diverse group of proteins. The C 15 and C 20 prenylated proteins have been identified by metabolic labeling with radioisotopic mevalonate, but many organisms do not incorporate this precursor readily. We have shown that the thioether bond of prenylated proteins can be cleaved by 2-naphthoxide to yield substituted naphthopyrans 2b and 2c. These products are readily resolved by HPLC due to their strongly absorbing chromophore. Thus, this reaction is suitable for qualitative identification of prenylated proteins. The results of model studies and a proposed reaction mechanism for the formation of 2b and 2c are discussed.