Inhibition of purified p21ras farnesyl: protein transferase by Cys-AAX tetrapeptides

Y Reiss, JL Goldstein, MC Seabra, PJ Casey… - Cell, 1990 - cell.com
Y Reiss, JL Goldstein, MC Seabra, PJ Casey, MS Brown
Cell, 1990cell.com
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-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 …
Summary
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-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~ 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.
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