The G protein α(s) subunit incorporates [3H]palmitic acid and mutation of cysteine-3 prevents this modification

M. Y. Degtyarev, Allen M. Spiegel, T. L.Z. Jones

Research output: Contribution to journalArticle

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Abstract

We investigated whether α(s) could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of α(s) and metabolically labeling with [3H]palmitate or [35S]methionine. Cells were separated into particulate and soluble fractions and immunoprecipitated with a specific peptide antibody. [3H]Palmitate was incorporated into both endogenous and transfected α(s). Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of α(s) with [3H]palmitate. Hydroxylamine treatment caused a release of the tritium radiolabel, demonstrating that the incorporation was through a thioester bond. The tritium radiolabel was base-labile and comigrated with [3H]palmitate on thin-layer chromatography. The third residue of the wild-type α(s) was mutated from a cysteine to an alanine by site-directed mutagenesis. This mutant was expressed in COS cells and localized to the particulate fraction as determined by immunoprecipitation of the [35S]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [3H]palmitate, indicating that this residue is crucial for the modification.

Original languageEnglish (US)
Pages (from-to)8057-8061
Number of pages5
JournalBiochemistry
Volume32
Issue number32
StatePublished - 1993
Externally publishedYes

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Palmitic Acid
Palmitates
Protein Subunits
GTP-Binding Proteins
Cysteine
Mutation
Tritium
COS Cells
Methionine
Thin layer chromatography
Mutagenesis
Hydroxylamine
Cycloheximide
Thin Layer Chromatography
Site-Directed Mutagenesis
Immunoprecipitation
Alanine
Labeling
Complementary DNA
Peptides

ASJC Scopus subject areas

  • Biochemistry

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The G protein α(s) subunit incorporates [3H]palmitic acid and mutation of cysteine-3 prevents this modification. / Degtyarev, M. Y.; Spiegel, Allen M.; Jones, T. L.Z.

In: Biochemistry, Vol. 32, No. 32, 1993, p. 8057-8061.

Research output: Contribution to journalArticle

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