High-performance liquid chromatographic assay for farnesyl-protein transferase activity with dabsylated peptide.
An HPLC assay for farnesyl-protein transferase activity using a dabsylated peptide is described. The substrates used were a synthetic dabsylated nonapeptide, N-dabsyl-L-serinyl-L-methioninyl-L-glycinyl-L-++ +leucinyl-L-prolinyl-L-cysteinyl- L-valinyl-L-valinyl-L-methionine, corresponding to the C-terminal peptide sequence of human N-Ras p21 without the N-terminal serine, and farnesyl diphosphate. The product was separated from the substrates on a reversed-phase C18 column, using gradient elution with acetonitrile (0.05% trifluoroacetic acid)-water (0.1% trifluoroacetic acid) and was detected at 436 nm. The addition of the farnesyl group to the peptide was confirmed by MS and NMR. Enzymatic reaction was ascertained from the dependences on time, on the protein of the enzyme source and on the substrates. The reaction was specifically inhibited by L-cysteinyl-L-valinyl-L-valinyl-L-methionine, the tetrapeptide corresponding to the "CAAX" motif. The limit of detection was 2 pmol per 100-microliters reaction mixture. The farnesyl-protein transferase activity can quantitatively be measured up to 200 micrograms cytosolic protein in human liver. This method provides a convenient and quantitative assay for crude materials, such as tissue homogenate from clinical samples, without the use of radioactive probes and large amounts of Ras protein.
Ishiguro
H
H
Second Department of Internal Medicine, Osaka University Medical School, Japan.
Kawata
S
S
Yamasaki
E
E
Matsuda
Y
Y
Fujii
S
S
Matsuzawa
Y
Y
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D017368:Protein Prenylation; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014166:Transferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives*
D010455:Peptides; D004124:p-Dimethylaminoazobenzene; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase