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 p osttranslational processing reactions-prenylation, endoproteolysis, and car boxymethylation. Two genes in yeast encoding Ca(1)a(2)X endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysi s of yeast Ras proteins. When proteolysis is blocked, localization of Ras2p to the outer membrane is impaired. The mislocalization of undermodified Ra s in the cell suggests that Rce1p is an attractive target for cancer therap eutics. A biotinylated, farnesylated Ca(1)a(2)X peptide [(1-N-biotinyl-(13- N-succinimidyl-(S-(E,E-farnesyl)-L-cysteinyl)-L-isoleucinyl-L-alanine))-4,7 ,10-trioxatridccanediamine] 1 containing a poly(ethylene glycol) linker was prepared by solid-phase synthesis for use in an assay for Ca(1)a(2)X endop rotease activity that relies on the strong affinity of avidin for biotin. T he peptide was radiolabeled in the penultimate step of the synthesis by cle avage of the biotinylated, farnesylated Ca(1)a(2) precursor from Kaiser's o xime resin with [C-14]-L-alanine methyl ester. [C-14]1 was a good substrate for yRce1p with K-M = 1.3 +/- 0.3 muM. Analysis of the carboxyl terminal p roducts by reverse phase HPLC confirmed that VLA was the only radioactive f ragment released upon incubation of [C-14]1 with a yeast membrane preparati on of recombinant yRce1p. The solid-phase methodology developed using Kaise r's benzophenone oxime resin to synthesize [C-14]1 should be generally appl icable for peptides containing sensitive side chains. In addition, introduc tion of the radiolabeled unit at the end of the synthesis mostly circumvent s problems associated with handling radioactive materials.