In vitro and cellular assays for palmitoyl acyltransferases using fluorescent lipidated peptides

Charles E. Ducker, Jeremiah M. Draper, Zuping Xia, Charles Smith

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Protein palmitoylation is emerging as an important post-translational modification in development as well as in the establishment and progression of diseases such as cancer. This chapter describes the use of fluorescent lipidated peptides to characterize palmitoyl acyltransferase (PAT) activities in vitro and in intact cells. The peptides mimic two motifs that are enzymatically palmitoylated, i.e. C-terminal farnesyl and N-terminal myristoyl sequences. These substrate peptides can be separated from the palmitoylated product peptides by reversed-phase HPLC, detected and quantified by the fluorescence of their NBD label. Through these methods, the activities of PATs toward these alternate substrates in isolated membranes or intact cells can be quantified. The in vitro assay has been used to characterize human PATs and to identify inhibitors of these enzymes. The cellular assay has been useful in elucidating the kinetics of protein palmitoylation by PATs in situ, and the sub-cellular distribution of the palmitoylated products.

Original languageEnglish (US)
Pages (from-to)166-170
Number of pages5
JournalMethods
Volume40
Issue number2
DOIs
StatePublished - Oct 1 2006

All Science Journal Classification (ASJC) codes

  • Molecular Biology

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