A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity

Amanda S. Varner, Mackenzie L. De Vos, Steffen P. Creaser, Blake R. Peterson, Charles D. Smith

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Although protein palmitoylation is essential for targeting many important signaling proteins to the plasma membrane, the mechanism by which palmitoylation occurs is uncharacterized, since the enzyme(s) responsible for this modification remain unidentified. To study palmitoyl acyl transferase (PAT) activity, we developed an in vitro palmitoylation (IVP) assay using a fluorescently labeled substrate peptide, mimicking the N-terminal palmitoylation motif of proteins such as non-receptor Src-related tyrosine kinases. The palmitoylated and non-palmitoylated forms of the peptide were resolved by reverse-phase HPLC and detected by fluorescence. The method was optimized for PAT activity using lysates from the MCF-7 and Hep-G2 human tumor cell lines. The PAT activity was inhibited by boiling, reducing the incubation temperature, or adding 10μM 2-bromopalmitate, a known palmitoylation inhibitor. This IVP assay provides the first method that is suitable to study all facets of the palmitoylation reaction, including peptide palmitoylation by PAT(s), depalmitoylation by thioesterases, and evaluation of potential palmitoylation inhibitors.

Original languageEnglish (US)
Pages (from-to)160-167
Number of pages8
JournalAnalytical Biochemistry
Volume308
Issue number1
DOIs
StatePublished - Sep 1 2002

Fingerprint

Lipoylation
Transferases
Fluorescence
Liquids
Peptides
Assays
Proteins
Cell membranes
Protein-Tyrosine Kinases
Boiling liquids
Tumors
Cells
Amino Acid Motifs
Substrates
Enzymes
src-Family Kinases
Tumor Cell Line
High Pressure Liquid Chromatography
Cell Membrane
Temperature

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Varner, Amanda S. ; De Vos, Mackenzie L. ; Creaser, Steffen P. ; Peterson, Blake R. ; Smith, Charles D. / A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity. In: Analytical Biochemistry. 2002 ; Vol. 308, No. 1. pp. 160-167.
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A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity. / Varner, Amanda S.; De Vos, Mackenzie L.; Creaser, Steffen P.; Peterson, Blake R.; Smith, Charles D.

In: Analytical Biochemistry, Vol. 308, No. 1, 01.09.2002, p. 160-167.

Research output: Contribution to journalArticle

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