Journal article
Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs
Biochemical journal, Vol.373(1), pp.91-99
07/01/2003
Handle:
https://hdl.handle.net/2376/113395
PMCID: PMC1223475
PMID: 12670300
Abstract
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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- Title
- Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs
- Creators
- Amanda S VARNER - Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.ACharles E DUCKER - Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.AZuping XIA - Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.AYan ZHUANG - Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.AMackenzie L DE VOS - Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.ACharles D SMITH - Department of Pharmacology, H078, Penn State College of Medicine, Hershey, PA 17033, U.S.A
- Publication Details
- Biochemical journal, Vol.373(1), pp.91-99
- Academic Unit
- Pharmacy and Pharmaceutical Sciences, College of
- Identifiers
- 99900547578201842
- Language
- English
- Resource Type
- Journal article