Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides.
We report the identification, purification, and characterization of a farnesyl:protein transferase that transfers the farnesyl moiety from farnesyl pyrophosphate to a cysteine in p21ras proteins. The enzyme was purified approximately 60,000-fold from rat brain cytosol through use of a chromatography step based on the enzyme's ability to bind to a hexapeptide containing the consensus sequence (Cys-AAX) for farnesylation. The purified enzyme migrated on gel filtration chromatography with an apparent molecular weight of 70,000-100,000. High resolution SDS-polyacrylamide gels showed two closely spaced approximately 50 kd protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as 4 residues that contained the Cys-AAX motif. These peptides acted as alternative substrates that competed with p21H-ras for farnesylation. Effective peptides included the COOH-terminal sequences of all known p21ras proteins as well as those of lamin A and B.
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Y
Y
Reiss
J L
JL
Goldstein
M C
MC
Seabra
P J
PJ
Casey
M S
MS
Brown
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D051381:Rats; D011919:Rats, Inbred Strains; D012717:Sesquiterpenes; D013329:Structure-Activity Relationship; D014166:Transferases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D009842:Oligopeptides; D011106:Polyisoprenyl Phosphates; D011518:Proto-Oncogene Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D011505:Protein-Tyrosine Kinases; D016283:Proto-Oncogene Proteins p21(ras)