TY - JOUR
T1 - Purification of ras farnesyl:Protein transferase
AU - Reiss, Yuval
AU - Seabra, Miguel C.
AU - Goldstein, Joseph L.
AU - Brown, Michael S.
N1 - Funding Information:
We thank our colleagues Sarah Stradley and Lila Gierasch for helpful discussions and synthesis of peptides. Richard Gibson and Debra Noble provided excellent technical assistance. This research was supported by grants from the National Institutes of Health (HL 20948), the Lucille P. Markey Charitable Trust, and the Perot Family Foundation. Y.R. is the recipient of a Chaim Weizmann postdoctoral fellowship award. M.C.S. is the recipient of a graduate fellowship from Fundacao Calouste Gulbenkian of Portugal and a Fullbright Scholarship.
PY - 1990/12
Y1 - 1990/12
N2 - We describe a method for the purification of farnesyl:protein transferase, an enzyme that transfers a farnesyl group from farnesyl pyrophosphate to a COOH-terminal cysteine in ras proteins, nuclear lamin B, and the γ subunit of bovine transducin. The enzyme is purified to homogeneity from rat brain cytosol through use of an affinity chromatography step based on the enzyme's ability to specifically bind to a hexapeptide containing the consensus sequence for farnesylation. The purification procedure is reproducible and enables the isolation of microgram amounts of purified enzyme from 50 rat brains. Two methods for assaying enzymatic activity are also described. One assay measures the transfer of [3H]farnesyl from [3H]farnesyl pyrophosphate to recombinant H-ras, and the other measures the transfer of [3H]farnesyl to a biotinylated peptide containing the Cys-AAX COOH-terminal sequence of K-rasB.
AB - We describe a method for the purification of farnesyl:protein transferase, an enzyme that transfers a farnesyl group from farnesyl pyrophosphate to a COOH-terminal cysteine in ras proteins, nuclear lamin B, and the γ subunit of bovine transducin. The enzyme is purified to homogeneity from rat brain cytosol through use of an affinity chromatography step based on the enzyme's ability to specifically bind to a hexapeptide containing the consensus sequence for farnesylation. The purification procedure is reproducible and enables the isolation of microgram amounts of purified enzyme from 50 rat brains. Two methods for assaying enzymatic activity are also described. One assay measures the transfer of [3H]farnesyl from [3H]farnesyl pyrophosphate to recombinant H-ras, and the other measures the transfer of [3H]farnesyl to a biotinylated peptide containing the Cys-AAX COOH-terminal sequence of K-rasB.
UR - http://www.scopus.com/inward/record.url?scp=0000504764&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0000504764&partnerID=8YFLogxK
U2 - 10.1016/S1046-2023(05)80323-8
DO - 10.1016/S1046-2023(05)80323-8
M3 - Article
AN - SCOPUS:0000504764
SN - 0076-6879
VL - 1
SP - 241
EP - 245
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - 3
ER -