Farnesyl-protein transferase and geranylgeranyl-protein transferase assays using phosphocellulose paper absorption.
Farnesyl-protein transferase catalyzes the reaction of farnesyl pyrophosphate and its acceptors to yield farnesyl protein and pyrophosphate. Geranylgeranyl-protein transferases are distinct enzymes that catalyze the reaction of geranylgeranyl pyrophosphate and their acceptors. We used tritiated isoprenoid pyrophosphate donors and synthetic peptide acceptors to measure enzyme activities. The peptide acceptors contained basic amino acid residues on the amino terminus of tetrapeptide substrate determinants specific for each enzyme. Following the incubation, portions of the reaction mixture were applied to numbered phosphocellulose paper strips that were immersed in 95% ethanol/75 mM phosphoric acid (1/1; v/v). Acid promoted binding of positively charged peptide substrates and products to the negatively charged paper, and alcohol eluted the radioactive prenyl groups. Paper strips were processed in the same container in batches for 40 min, and radioactivity adsorbed to the strips was then measured by liquid scintillation spectrometry. The use of peptides makes the expression and purification of recombinant substrates in bacteria unnecessary. However, most proteins bind quantitatively to phosphocellulose at acidic pH, and the washing procedure developed for peptide substrates is applicable for measuring prenyltransferase activities with recombinant Ras proteins as acceptor.
Roskoski
R
R
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119.
Ritchie
P
P
Gahn
L G
LG
D000042:Absorption; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D007700:Kinetics; D008969:Molecular Sequence Data; D010209:Paper; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D013379:Substrate Specificity; D014166:Transferases / Q000032:analysis*
D011994:Recombinant Proteins; D002482:Cellulose; C008881:phosphocellulose; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C064911:p21(ras) farnesyl-protein transferase