Solid-phase synthesis of a radiolabeled, biotinylated, and farnesylated Ca(1)a(2)X peptide substrate for Ras- and a-mating factor converting enzyme.
Eukaryotic proteins with carboxyl-terminal Ca(1)a(2) motifs undergo three posttranslational processing reactions--prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding Ca(1)a(2)X endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, localization of Ras2p to the outer membrane is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. A biotinylated, farnesylated Ca(1)a(2)X peptide [(1-N-biotinyl-(13-N-succinimidyl-(S-(E,E-farnesyl)-L-cysteinyl)-L-valinyl-L-isoleucinyl-L-alanine))-4,7,10-trioxatridecanediamine] 1 containing a poly(ethylene glycol) linker was prepared by solid-phase synthesis for use in an assay for Ca(1)a(2)X endoprotease activity that relies on the strong affinity of avidin for biotin. The peptide was radiolabeled in the penultimate step of the synthesis by cleavage of the biotinylated, farnesylated Ca(1)a(2) precursor from Kaiser's oxime resin with [(14)C]-L-alanine methyl ester. [(14)C]1 was a good substrate for yRce1p with K(M) = 1.3 +/- 0.3 microM. Analysis of the carboxyl terminal products by reverse phase HPLC confirmed that VIA was the only radioactive fragment released upon incubation of [(14)C]1 with a yeast membrane preparation of recombinant yRce1p. The solid-phase methodology developed using Kaiser's benzophenone oxime resin to synthesize [(14)C]1 should be generally applicable for peptides containing sensitive side chains. In addition, introduction of the radiolabeled unit at the end of the synthesis mostly circumvents problems associated with handling radioactive materials.
Dolence
E K
EK
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112-0850, USA.
Dolence
J M
JM
Poulter
C D
CD
D019921:Biotinylation; D010450:Endopeptidases / Q000737:chemistry*; D007202:Indicators and Reagents; D007553:Isotope Labeling; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry*; D008565:Membrane Proteins; D008666:Metalloendopeptidases / Q000737:chemistry*; D009842:Oligopeptides / Q000138:chemical synthesis*; D010675:Pheromones; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins / Q000737:chemistry*
C424743:(1-N-biotinyl-(13-N-succinimidyl-((E,E-farnesyl)-cysteinyl)-valinyl-isoleucinyl-alanine))-4,7,10-trioxatridecanediamine; D007202:Indicators and Reagents; D008074:Lipoproteins; C095063:MFA2 protein, S cerevisiae; D008565:Membrane Proteins; D009842:Oligopeptides; D010675:Pheromones; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; D043484:Proprotein Convertases; C105193:RCE1 protein, S cerevisiae; D008666:Metalloendopeptidases; C491251:STE24 protein, S cerevisiae; D018631:ras Proteins