The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity towards the myristoylated substrate peptide but not the farnesylated peptide, while ras-transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the Km values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the Km for MyrGCK(NBD) was 5-fold lower than the Km for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC50 values of approx. 4 and 1.3 μM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N-ethylmaleimide and thermal denaturation. Overall, these data demonstrate that the enzymic palmitoylation of farnesyl- and myristoyl-containing peptide substrates can be differentiated, suggesting that multiple motif-specific PATs exist.
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Research Article|
July 01 2003
Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs
Amanda S. VARNER;
Amanda S. VARNER
Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A.
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Charles E. DUCKER;
Charles E. DUCKER
Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A.
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Zuping XIA;
Zuping XIA
Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A.
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Yan ZHUANG;
Yan ZHUANG
Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A.
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Mackenzie L. DE VOS;
Mackenzie L. DE VOS
Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A.
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Charles D. SMITH
Charles D. SMITH
1
Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A.
1To whom correspondence should be addressed (e-mail cdsmith@psu.edu).
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Publisher: Portland Press Ltd
Received:
October 11 2002
Revision Received:
March 07 2003
Accepted:
April 01 2003
Accepted Manuscript online:
April 01 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 373 (1): 91–99.
Article history
Received:
October 11 2002
Revision Received:
March 07 2003
Accepted:
April 01 2003
Accepted Manuscript online:
April 01 2003
Citation
Amanda S. VARNER, Charles E. DUCKER, Zuping XIA, Yan ZHUANG, Mackenzie L. DE VOS, Charles D. SMITH; Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs. Biochem J 1 July 2003; 373 (1): 91–99. doi: https://doi.org/10.1042/bj20021598
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