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Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase

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Abstract

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 μM. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 & mgr;M. The E. coli expressed sNMT was homogenous and showed enzyme activity.

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Authors and Affiliations

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

    Rajala V.S. Raju, Rakesh Kakkar & Rajendra K. Sharma

  2. Plant Biotechnology Institute, National Research Council, Saskatoon, Saskatchewan, Canada

    Raju S.S. Datla

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  1. Rajala V.S. Raju
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  2. Raju S.S. Datla
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  3. Rakesh Kakkar
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  4. Rajendra K. Sharma
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Raju, R.V., Datla, R.S., Kakkar, R. et al. Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase. Mol Cell Biochem 189, 91–97 (1998). https://doi.org/10.1023/A:1006861417562

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