A RADIOMETRIC ASSAY FOR RAS-PROCESSING PEPTIDASE USING AN ENZYMATICALLY RADIOLABELED PEPTIDE

A simple and sensitive radiometric assay for the peptidase involved in the post-translational processing of p21(ras) proteins at the carboxy -terminal Cys-aliphatic-aliphatic-any amino acid (CAAX) motif is descr ibed. An isoprenylated tetrapeptide substrate, N-acetyl-S-[H-3]farnesy l-Cys-Val-Ile-Ser-OH (22-27 Ci/mmol), was synthesized from N-acetyl-Cy s-Val-Ile-Ser-OH and commercial [H-3]farnesyl pyrophosphate via farnes yltransferase. The isoprenylated tetrapeptide was then used at a conce ntration (0.3 mu M) well below K-m (6 mu M) in assays with a microsoma l preparation of Ras-processing peptidase from bovine liver. Under ass ay conditions, the peptidase reaction followed first order kinetics wi th respect to the substrate, allowing the IC50 values for alternative substrates and inhibitors to approximate K-m and K-i values, respectiv ely. In a further simplification, substrate and N-acetyl-S-[H-3]farnes yl-Cys-OH product were separated by thin-layer chromatography on silic a gel plates using chloroform:acetic acid:methanol:acetone (60:5:10:20 , v/v) as solvents. The assay does not require costly, specialized equ ipment and provides easy means for screening potential substrates and inhibitors of Ras-processing peptidase. (C) 1994 Academic Press, Inc.