A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity.
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 10 microM 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.
Varner
Amanda S
AS
Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA.
De Vos
Mackenzie L
ML
Creaser
Steffen P
SP
Peterson
Blake R
BR
Smith
Charles D
CD
D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D008954:Models, Biological; D009227:Myristic Acids / Q000378:metabolism; D010168:Palmitates / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014407:Tumor Cells, Cultured; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
D009227:Myristic Acids; D010168:Palmitates; C022776:2-bromopalmitate; D019308:Palmitic Acid; D000217:Acyltransferases; C105599:HHAT protein, human; D019061:src-Family Kinases