Abstract
We describe methods that allow for the identification of prenylated and methyl-esterified carboxyl-terminal cysteine residues in proteins radiolabeled in vitro and in vivo . Proteins isotopically labeled in the 15-carbon farnesyl or 20-carbon geranylgeranyl prenyl group, the cysteine residue, or the methyl group are subjected to complete enzymatic hydrolysis under conditions that preserve the methyl ester. The products of this reaction are mixed with synthetic standards of farnesylcysteine, farnesylcysteine methyl ester, geranylgeranylcysteine, and geranylgeranylcysteine methyl ester and are fractionated by reverse-phase HPLC. Each of these compounds is readily resolved, and the product identification can be confirmed using normal-phase thin-layer chromatography. Further confirmation of the linkage of the methyl group to the α -carboxyl group of a carboxyl-terminal cysteine residue can be obtained by identifying radiolabeled cysteic acid methyl ester derived from performic acid-oxidized samples of digests of cysteine or methyl ester labeled material. Cysteic acid methyl ester can be identified by amino acid analysis on cation-exchange resin as well as by thin-layer chromatography. We describe synthetic methods for preparing the farnesyl- and geranylgeranylcysteine derivatives as standards, as well as general methods for detecting radiolabeled methyl esters.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.