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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 metal ions on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of (a) Ca2+, (b) CaM, (c) Mn2+, (d) Mg2+, and (e) Zn2+  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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fig4: Effect of metal ions on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of (a) Ca2+, (b) CaM, (c) Mn2+, (d) Mg2+, and (e) Zn2+ 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: We examined the effect of various divalent cations on NMT2. The metal ion Ca2+ had stimulatory effects on NMT2 activity at lower concentration (0.25 mM) while higher concentration showed inhibition of enzyme activity and 40% of the NMT2 enzyme activity inhibited at 5 mM concentration (Figure 4(a)). The myristoylated MARCKS, a major in vivo substrate of protein kinase C, interacts with plasma membranes in a phosphorylation-, myristoylation-, and calmodulin-dependent manner [28]. Therefore, we interested to examine the effect of calmodulin (CaM) on NMT2 activity and observed that there was no significant effect of CaM on NMT2 activity (Figure 4(b)). We observed a 50% loss of enzyme activity by Mn2+ at 5 mM concentration. Interestingly, Zn2+ showed maximum inhibition compared to other divalent cations tested and 80% of the enzyme activity was abolished at 5 mM concentration (Figures 4(c) and 4(e)). The metal ions Mg2+ had negligible effects on NMT2 activity, indicating that the enzyme does not require these divalent cations for its activity (Figure 4(d)). The metal ions Mg2+, Ca2+, and Mn2+ had negligible effects on Arabidopsis NMT1 activity, indicating that the enzyme does not require these divalent cations for its activity [36].


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

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

Effect of metal ions on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of (a) Ca2+, (b) CaM, (c) Mn2+, (d) Mg2+, and (e) Zn2+  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

fig4: Effect of metal ions on bovine brain NMT2 activity. Bovine brain NMT2 (1.0 μg/assay) activity was determined in the presence of carrying concentrations of (a) Ca2+, (b) CaM, (c) Mn2+, (d) Mg2+, and (e) Zn2+ 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: We examined the effect of various divalent cations on NMT2. The metal ion Ca2+ had stimulatory effects on NMT2 activity at lower concentration (0.25 mM) while higher concentration showed inhibition of enzyme activity and 40% of the NMT2 enzyme activity inhibited at 5 mM concentration (Figure 4(a)). The myristoylated MARCKS, a major in vivo substrate of protein kinase C, interacts with plasma membranes in a phosphorylation-, myristoylation-, and calmodulin-dependent manner [28]. Therefore, we interested to examine the effect of calmodulin (CaM) on NMT2 activity and observed that there was no significant effect of CaM on NMT2 activity (Figure 4(b)). We observed a 50% loss of enzyme activity by Mn2+ at 5 mM concentration. Interestingly, Zn2+ showed maximum inhibition compared to other divalent cations tested and 80% of the enzyme activity was abolished at 5 mM concentration (Figures 4(c) and 4(e)). The metal ions Mg2+ had negligible effects on NMT2 activity, indicating that the enzyme does not require these divalent cations for its activity (Figure 4(d)). The metal ions Mg2+, Ca2+, and Mn2+ had negligible effects on Arabidopsis NMT1 activity, indicating that the enzyme does not require these divalent cations for its activity [36].

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