« Previous AbstractSex-Pairing Pheromones in Three Sympatric Neotropical Termite Species (Termitidae: Syntermitinae)    Next AbstractQualitative analyses of less-volatile organic molecules from female skin scents by comprehensive two dimensional gas chromatography-time of flight mass spectrometry »

Bioconjug Chem

Title:		"Solid-phase synthesis of a radiolabeled, biotinylated, and farnesylated Ca(1)a(2)X peptide substrate for Ras- and a-mating factor converting enzyme"
Author(s):		Dolence EK; Dolence JM; Poulter CD;
Address:		"Department of Chemistry, University of Utah, Salt Lake City, Utah 84112-0850, USA"
Journal Title:		Bioconjug Chem
Year:		2001
Volume:		12
Issue:		1
Page Number:		35 - 43
DOI:		10.1021/bc000036g
ISSN/ISBN:		1043-1802 (Print) 1043-1802 (Linking)
Abstract:		"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"
Keywords:		Biotinylation Endopeptidases/*chemistry Indicators and Reagents Isotope Labeling Kinetics Lipoproteins/*chemistry *Membrane Proteins Metalloendopeptidases/*chemistry Oligopeptides/*chemical synthesis Pheromones Proprotein Convertases Recombinant Proteins/;
Notes:		"MedlineDolence, E K Dolence, J M Poulter, C D eng Research Support, Non-U.S. Gov't 2001/02/15 Bioconjug Chem. 2001 Jan-Feb; 12(1):35-43. doi: 10.1021/bc000036g"