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Biochemical characterization of bovine brain myristoyl-CoA:protein N-myristoyltransferase type 2.

Selvakumar P, Lakshmikuttyamma A, Sharma RK - J. Biomed. Biotechnol. (2009)

Bottom Line: Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities.The metal ion Ca(2+) had stimulatory effects on NMT2 activity while Mn(2+) and Zn(2+) inhibited the enzyme activity.In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, College of Medicine and Health Research Division, Saskatchewan Cancer Agency, University of Saskatchewan, SK, Canada.

ABSTRACT
Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) from Bos tarus brain. The open reading frame codes for a 410-amino-acid protein and overexpressed in Escherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ion Ca(2+) had stimulatory effects on NMT2 activity while Mn(2+) and Zn(2+) inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

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Related in: MedlinePlus

Effect of SDS on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of SDS (0–10 mM) using cAMP-dependent protein kinase derived peptide substrate (1.0 mM). The reactions were initiated by the addition of 0.27 μM[3H] myristoyl CoA and incubated at 30°C for 30 minutes. The data presented are representative of at least three separate experiments.
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fig5: Effect of SDS on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of SDS (0–10 mM) using cAMP-dependent protein kinase derived peptide substrate (1.0 mM). The reactions were initiated by the addition of 0.27 μM[3H] myristoyl CoA and incubated at 30°C for 30 minutes. The data presented are representative of at least three separate experiments.

Mentions: Another interesting finding observed in this study was that NMT2 was found to be activated 2.5 folds with SDS. The optimum concentration was found to be 3.0 mM (Figure 5). These results suggest that a mild concentration of detergent could open the structure of NMT2, thereby allowing NMT2 to become highly active. However at higher concentrations of SDS, NMT2 activity was totally inactivated, suggesting that there is an optimum level of structural changes in NMT2. Results also suggest that some thiol group(s) in NMT2 could be masked inside the protein. In the presence of SDS, these thiol group(s) could be unmasked and thereby reactive. Human NMT1 was found to be activated several folds with SDS and optimum concentration was found to be 1.73 mM [37]. It has been reported that in aldolase, one thiol per subunit reacts readily, but an additional three groups react if a small amount of detergent is added [38]. Human NMT has been shown to be susceptible towards N-5,5′ dithiobis (2-nitro-benzoic acid) (DTNB) and iodoacetamide, suggesting the implication of cysteine requirement for the enzyme catalysis [39]. Comparison of four yeasts and one human NMT protein sequences revealed that 2 cysteine residues at amino acids 169 and 214 in human NMT and 172 and 217 in yeast NMT were highly conserved [40, 41]. Bovine spleen NMT has been shown to be activated several folds with SDS [42].


Biochemical characterization of bovine brain myristoyl-CoA:protein N-myristoyltransferase type 2.

Selvakumar P, Lakshmikuttyamma A, Sharma RK - J. Biomed. Biotechnol. (2009)

Effect of SDS on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of SDS (0–10 mM) using cAMP-dependent protein kinase derived peptide substrate (1.0 mM). The reactions were initiated by the addition of 0.27 μM[3H] myristoyl CoA and incubated at 30°C for 30 minutes. The data presented are representative of at least three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2737134&req=5

fig5: Effect of SDS on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of SDS (0–10 mM) using cAMP-dependent protein kinase derived peptide substrate (1.0 mM). The reactions were initiated by the addition of 0.27 μM[3H] myristoyl CoA and incubated at 30°C for 30 minutes. The data presented are representative of at least three separate experiments.
Mentions: Another interesting finding observed in this study was that NMT2 was found to be activated 2.5 folds with SDS. The optimum concentration was found to be 3.0 mM (Figure 5). These results suggest that a mild concentration of detergent could open the structure of NMT2, thereby allowing NMT2 to become highly active. However at higher concentrations of SDS, NMT2 activity was totally inactivated, suggesting that there is an optimum level of structural changes in NMT2. Results also suggest that some thiol group(s) in NMT2 could be masked inside the protein. In the presence of SDS, these thiol group(s) could be unmasked and thereby reactive. Human NMT1 was found to be activated several folds with SDS and optimum concentration was found to be 1.73 mM [37]. It has been reported that in aldolase, one thiol per subunit reacts readily, but an additional three groups react if a small amount of detergent is added [38]. Human NMT has been shown to be susceptible towards N-5,5′ dithiobis (2-nitro-benzoic acid) (DTNB) and iodoacetamide, suggesting the implication of cysteine requirement for the enzyme catalysis [39]. Comparison of four yeasts and one human NMT protein sequences revealed that 2 cysteine residues at amino acids 169 and 214 in human NMT and 172 and 217 in yeast NMT were highly conserved [40, 41]. Bovine spleen NMT has been shown to be activated several folds with SDS [42].

Bottom Line: Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities.The metal ion Ca(2+) had stimulatory effects on NMT2 activity while Mn(2+) and Zn(2+) inhibited the enzyme activity.In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, College of Medicine and Health Research Division, Saskatchewan Cancer Agency, University of Saskatchewan, SK, Canada.

ABSTRACT
Protein N-myristoylation is a lipidic modification which refers to the covalent attachment of myristate, a 14-carbon saturated fatty acid, to the N-terminal glycine residue of a number of mammalian, viral, and fungal proteins. In this paper, we have cloned the gene coding for myristoyl-CoA:protein N-myristoyltransferase (NMT) from Bos tarus brain. The open reading frame codes for a 410-amino-acid protein and overexpressed in Escherichia coli. Kinetic studies suggested that bovine brain NMT2 and human NMT1 show significant differences in their peptide substrate specificities. The metal ion Ca(2+) had stimulatory effects on NMT2 activity while Mn(2+) and Zn(2+) inhibited the enzyme activity. In addition, NMT2 activity was inhibited by various organic solvents and other detergents while NMT1 had a stimulatory effect. Biochemical characterization suggested that both forms of NMT have unique characteristics. Further analysis towards functional role NMT2 will lead the development of therapeutic target for the progression of various diseases such as cancer, cardiovascular diseases, and neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus