Purification of ras farnesyl:Protein transferase

Yuval Reiss; Miguel C. Seabra; Joseph L. Goldstein; Michael S. Brown

doi:10.1016/S1046-2023(05)80323-8

Purification of ras farnesyl:Protein transferase

Yuval Reiss, Miguel C. Seabra, Joseph L. Goldstein, Michael S. Brown

Research output: Contribution to journal › Article › peer-review

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Abstract

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.

Original language	English (US)
Pages (from-to)	241-245
Number of pages	5
Journal	Methods
Volume	1
Issue number	3
DOIs	https://doi.org/10.1016/S1046-2023(05)80323-8
State	Published - Dec 1990

ASJC Scopus subject areas

Molecular Biology
General Biochemistry, Genetics and Molecular Biology

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@article{e576ebda586b4096b34aa52e817d0ffe,

title = "Purification of ras farnesyl:Protein transferase",

abstract = "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.",

author = "Yuval Reiss and Seabra, {Miguel C.} and Goldstein, {Joseph L.} and Brown, {Michael S.}",

note = "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.",

year = "1990",

month = dec,

doi = "10.1016/S1046-2023(05)80323-8",

language = "English (US)",

volume = "1",

pages = "241--245",

journal = "Methods",

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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.

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Purification of ras farnesyl:Protein transferase

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