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.
Epstein
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WW
Department of Chemistry, University of Utah, Salt Lake City 84108.
Lever
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Leining
L M
LM
Bruenger
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Rilling
H C
HC
D016475:3T3 Cells / Q000737:chemistry; D000818:Animals; D016466:CHO Cells / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D004224:Diterpenes / Q000032:analysis* / Q000737:chemistry; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D008798:Mevalonic Acid / Q000378:metabolism; D051379:Mice; D009284:Naphthols / Q000737:chemistry; D011499:Protein Processing, Post-Translational
D004224:Diterpenes; D009284:Naphthols; C065977:geranylgeranylcysteine; C057785:S-farnesylcysteine; D003545:Cysteine; C028405:2-naphthol; D008798:Mevalonic Acid