Document Detail


A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity.
MedLine Citation:
PMID:  12234477     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Amanda S Varner; Mackenzie L De Vos; Steffen P Creaser; Blake R Peterson; Charles D Smith
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Analytical biochemistry     Volume:  308     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2002 Sep 
Date Detail:
Created Date:  2002-09-17     Completed Date:  2003-03-20     Revised Date:  2010-09-17    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  160-7     Citation Subset:  IM    
Affiliation:
Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA.
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MeSH Terms
Descriptor/Qualifier:
Acylation
Acyltransferases / chemistry,  metabolism*
Chromatography, High Pressure Liquid / methods*
Fluorescence
Hot Temperature
Humans
Models, Biological
Myristic Acids / metabolism
Palmitates / pharmacology
Palmitic Acid / metabolism
Subcellular Fractions / enzymology
Tumor Cells, Cultured
src-Family Kinases / chemistry,  metabolism
Grant Support
ID/Acronym/Agency:
R01 CA 75248/CA/NCI NIH HHS; R01 CA 83831/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Myristic Acids; 0/Palmitates; 18263-25-7/2-bromopalmitate; 57-10-3/Palmitic Acid; EC 2.3.-/Acyltransferases; EC 2.3.1.-/HHAT protein, human; EC 2.7.10.2/src-Family Kinases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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