FARNESYL-PROTEIN TRANSFERASE AND GERANYLGERANYL-PROTEIN TRANSFERASE ASSAYS USING PHOSPHOCELLULOSE PAPER ABSORPTION

Farnesyl-protein transferase catalyzes the reaction of farnesyl pyroph osphate and its accepters to yield farnesyl protein and pyrophosphate. Geranylgeranyl-protein transferases are distinct enzymes that catalyz e the reaction of geranylgeranyl pyrophosphate and their accepters. We used tritiated isoprenoid pyrophosphate donors and synthetic peptide accepters to measure enzyme activities. The peptide accepters containe d basic amino acid residues on the amino terminus of tetrapeptide subs trate determinants specific for each enzyme. Following the incubation, portions of the reaction mixture were applied to numbered phosphocell ulose paper strips that were immersed in 95% ethanol/75 mM phosphoric acid (1/1; v/v). Acid promoted binding of positively charged peptide s ubstrates and products to the negatively charged paper, and alcohol el uted the radioactive prenyl groups. Paper strips were processed in the same container in batches for 40 min, and radioactivity adsorbed to t he strips was then measured by liquid scintillation spectrometry. The use of peptides makes the expression and purification of recombinant s ubstrates in bacteria unnecessary. However, most proteins bind quantit atively to phosphocellulose at acidic pH, and the washing procedure de veloped for peptide substrates is applicable for measuring prenyltrans ferase activities with recombinant Pas proteins as acceptor. (C) 1994 Academic Press, Inc.