Recombinant bovine spleen myristoyl CoA: protein N-myristoyltransferase

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...

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Bibliographic Details
Published in:Molecular and cellular biochemistry 1998-12, Vol.189 (1-2), p.91-97
Main Authors: Raju, R V, Datla, R S, Kakkar, R, Sharma, R K
Format: Article
Language:English
Subjects:
Acyltransferases - biosynthesis
Acyltransferases - chemistry
Acyltransferases - genetics
Amino Acid Sequence
Animals
Base Sequence
Cattle
E coli
Enzymatic activity
Enzymes
Escherichia coli - genetics
Kinases
Liquid chromatography
Molecular Sequence Data
Molecular Weight
Nickel - pharmacology
Proteins
Recombinant Proteins - biosynthesis
Spleen
Spleen - enzymology
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Summary: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 microM. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 microM. The E. coli expressed sNMT was homogenous and showed enzyme activity.
ISSN:0300-8177
1573-4919
DOI:10.1023/A:1006861417562