Solid-Phase Synthesis of a Radiolabeled, Biotinylated, and Farnesylated Ca1a2X Peptide Substrate for Ras- and a-Mating Factor Converting Enzyme
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- 作者：E. Kurt Dolence ; Julia M. Dolence ; and C. Dale Poulter
- 刊名：Bioconjugate Chemistry
- 出版年：2001
- 出版时间：January 2001
- 年：2001
- 卷：12
- 期：1
- 页码：35 - 43
- 全文大小：100K
- 年卷期：v.12,no.1(January 2001)
- ISSN：1520-4812
文摘
Eukaryotic proteins with carboxyl-terminal Ca1a2 motifs undergo three posttranslational processingreactions-prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding Ca1a2Xendoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis ofyeast Ras proteins. When proteolysis is blocked, localization of Ras2p to the outer membrane isimpaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. A biotinylated, farnesylated Ca1a2X 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 synthesisfor use in an assay for Ca1a2X endoprotease activity that relies on the strong affinity of avidin forbiotin. The peptide was radiolabeled in the penultimate step of the synthesis by cleavage of thebiotinylated, farnesylated Ca1a2 precursor from Kaiser's oxime resin with [14C]-L-alanine methyl ester.[14C]1 was a good substrate for yRce1p with KM = 1.3 ± 0.3 
M. Analysis of the carboxyl terminalproducts by reverse phase HPLC confirmed that VIA was the only radioactive fragment released uponincubation of [14C]1 with a yeast membrane preparation of recombinant yRce1p. The solid-phasemethodology developed using Kaiser's benzophenone oxime resin to synthesize [14C]1 should begenerally applicable for peptides containing sensitive side chains. In addition, introduction of theradiolabeled unit at the end of the synthesis mostly circumvents problems associated with handlingradioactive materials.
M. Analysis of the carboxyl terminalproducts by reverse phase HPLC confirmed that VIA was the only radioactive fragment released uponincubation of [14C]1 with a yeast membrane preparation of recombinant yRce1p. The solid-phasemethodology developed using Kaiser's benzophenone oxime resin to synthesize [14C]1 should begenerally applicable for peptides containing sensitive side chains. In addition, introduction of theradiolabeled unit at the end of the synthesis mostly circumvents problems associated with handlingradioactive materials.