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Preparation and Assay of Myristoyl-CoA:Protein N-Myristoyltransferase

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Protein Lipidation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 116))

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Abstract

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational transfer of myristate from myristoyl-CoA to the amino-terminal glycine residue of a number of cellular, viral, fungal, and oncoproteins (1–5). These proteins include the catalytic subunit of cAMP-dependent protein kinase, various tyrosine kinases (including pp60src, pp60yes, pp56lck, pp59fyn/syn, and cAbl), the β-subunit of calmodulin-dependent protein phosphatase (calcineurin), the myristoylated alanine rich C kinase substrate, the α-subunit of several G-proteins, and several ARF proteins involved in ADP ribosylation (1–5). A significant problem with the purification and characterization of NMT concerns the available assay procedures. The wide availability and use of peptides allow for convenient substrates. However, the existing procedures to isolate and analyze the reaction products of myristoylation, such as reverse-phase HPLC, are expensive and time-consuming (6,7). Ion-exchange column chromatography, differential solubilization, adsorption of myristoyl-CoA to acidic alumina, and ion-exchange exclusion of [3H]myristoyl peptides have been utilized as assay methods are faster and more convenient than reverse-phase HPLC (8–10). However, these procedures are still limited with respect to the number of assays that can conveniently be performed. Here we described a new rapid, reliable, and inexpensive NMT assay based on the binding of [3H]myristoyl-peptide to a P81 phosphocellulose paper matrix (11).

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Authors and Affiliations

  1. Department of Pathology and Saskatoon Cancer Center, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    Rajala V. S. Raju & Rajendra K. Sharma

Authors
  1. Rajala V. S. Raju
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  2. Rajendra K. Sharma
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Editor information

Editors and Affiliations

  1. University of Washington, Seattle, WA

    Michael H. Gelb

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© 1998 Humana Press Inc.

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Raju, R.V.S., Sharma, R.K. (1998). Preparation and Assay of Myristoyl-CoA:Protein N-Myristoyltransferase. In: Gelb, M.H. (eds) Protein Lipidation Protocols. Methods in Molecular Biology, vol 116. Humana Press. https://doi.org/10.1385/1-59259-264-3:193

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  • DOI: https://doi.org/10.1385/1-59259-264-3:193

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-534-8

  • Online ISBN: 978-1-59259-264-7

  • eBook Packages: Springer Protocols

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