Biophys. Formation of a doubly-palmitoylated GAP-peptide product was confirmed by mass spectroscopy. The GAP-peptide substrate was separated from your palmitoylated peptide product in under 7 moments by MEKC. The rate of palmitoylation with respect to reaction time, GAP-peptide concentration, pH, and inhibitor concentration were also examined. This capillary electrophoresis-based assay for monitoring palmitoylation has applications in biochemical studies of acyltransferases and thioesterases as well as in the screening of acyltransferase and thioesterase inhibitors for drug development. a thioester bond to a cysteine in a protein. This dynamic and reversible modification increases Mogroside III the hydrophobicity of proteins, thereby increasing interactions with other hydrophobic moieties, such as the plasma membrane. Palmitoylation cycling plays a role in cell signaling Mogroside III by promoting the movement of proteins to different sites of action within the cell[1C9]. Palmitoylation is also implicated in the regulation of protein trafficking[10C14], as well as promoting protein-protein interactions[15,16] and modulating enzyme activity[17C19]. For example, palmitoylation of the growth associated protein, GAP-43, helps to direct the protein to the plasma membrane, where it is involved in neuronal growth and distributing, particularly the extension Mogroside III and branching of Mogroside III neuronal axon suggestions, or growth cones[20C23]. Space-43 is usually palmitoylated during axonal growth cone extension and branching, but is not palmitoylated during growth cone maturation[24,25]. Interestingly, GAP-43 protein expression in axons remains unchanged between growth and the final development of mature synapses. Palmitoylation, therefore, is the switch between axon growth and maturation, rather than complete protein expression. In the case of neuronal growth and Space-43, palmitoylation functions as an efficient cellular mechanism to control the construction and remodeling of a system as dynamic PDGFRA and plastic as a neuronal synapse in the developing brain. Palmitoylation of proteins and peptides is commonly monitored by isotope radiolabeling techniques. Protein or peptide substrates are incubated with tritiated palmitoyl coenzyme A ([3H]palmitoyl-CoA) with or without enzymes. The transfer of the [3H]palmitoyl moiety onto the substrate is usually monitored using SDS-PAGE separations and detected by gel staining techniques. While this technique is usually widely used, it has limitations. Radiolabeling is usually laborious and sample rigorous. The radioactive palmitoyl-CoA and the generated radioactive waste are expensive. The time to perform the full assay requires at least one week. Unfortunately, this technique is usually qualitative, limiting its use in kinetic studies. In addition, because only the palmitoyl group is usually radiolabeled, this technique cannot be used to quantitatively study the dynamic, and equally important, de-palmitoylation event. Recently, engineered peptides, selected for their structural similarity to native palmitoylated proteins, have been synthesized and used to study palmitoylation using high performance liquid chromatography (HPLC)[28C30]. Fluorescently-labeled Mogroside III tripeptide substrates representing the palmitoylation motif of ras proteins were used to characterize the activity of palmitoylation enzymes, known as acyltransferases, extracted from numerous malignancy cell lines. Using low-retention columns (HPLC-C4) and fluorescence detection, a palmitoylated ras tripeptide substrate was separated from non-palmitoylated substrate. Monitoring palmitoylation by HPLC offers the benefits of lower sample volumes (L) and quicker analysis time (several hours); however, HPLC utilizes large volumes of solvent with the attendant costs of waste disposal and the sample size remains too large for assays in which only small amounts of biologic reagents are available. Relative to HPLC, capillary electrophoresis (CE) has many advantages including very small samples sizes, ranging from pL to nL, detection limits as low as zeptomoles, and separation times of seconds to minutes, which are ideal conditions for monitoring dynamic chemical and biological processes in single cells or small groups of cells . CE-based separation of fluorescently-labeled peptides has been used to develop novel biochemical assays for monitoring and measuring the dynamics of different post-translational modifications, including phosphorylation[32C34], S-nitrosylation and farnesylation[36C38]. Fuorescently-labeled peptides, as opposed to proteins, are easier to weight into cells, can be designed to react with.
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