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 p21ras proteins at the carboxy-terminal Cys-aliphatic-aliphatic--any amino acid (CAAX) motif is described. An isoprenylated tetrapeptide substrate, N-acetyl-S-[3H]farnesyl-Cys-Val-Ile-Ser-OH (22-27 Ci/mmol), was synthesized from N-acetyl-Cys-Val-Ile-Ser-OH and commercial [3H]farnesyl pyrophosphate via farnesyltransferase. The isoprenylated tetrapeptide was then used at a concentration (0.3 microM) well below Km (6 microM) in assays with a microsomal preparation of Ras-processing peptidase from bovine liver. Under assay conditions, the peptidase reaction followed first order kinetics with respect to the substrate, allowing the IC50 values for alternative substrates and inhibitors to approximate Km and Ki values, respectively. In a further simplification, substrate and N-acetyl-S-[3H]farnesyl-Cys-OH product were separated by thin-layer chromatography on silica gel plates using chloroform:acetic acid:methanol:acetone (60:5:10:20, v/v) as solvents. The assay does not require costly, specialized equipment and provides easy means for screening potential substrates and inhibitors of Ras-processing peptidase.
Georgopapadakou
N H
NH
Department of Oncology, Roche Research Center, Nutley, New Jersey 07110-1199.
Hall
C C
CC
Lambros
T
T
Liu
W
W
Watkins
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism*; D006868:Hydrolysis; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis*; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011874:Radiometry / Q000379:methods; D012680:Sensitivity and Specificity; D014316:Tritium
D009842:Oligopeptides; D011480:Protease Inhibitors; D014316:Tritium; C089129:N-acetyl-S-farnesyl-cysteinyl-valyl-isoleucyl-serine; D010450:Endopeptidases; D016283:Proto-Oncogene Proteins p21(ras)