Limits...
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.

Show MeSH

Related in: MedlinePlus

SDS-PAGE and Western blot analysis of bovine brain NMT2. Thirty microgram of proteins was loaded onto (a) SDS-PAGE; lane 1, crude cell lysate; lane 2, purified E. coli  expressed bovine brain NMT2. (b) Purified E. coli  expressed bovine brain NMT2 (thirty microgram) immunoblotted with monoclonal anti-NMT2 (1 : 1000 dilution) as described in Section 2.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2737134&req=5

fig3: SDS-PAGE and Western blot analysis of bovine brain NMT2. Thirty microgram of proteins was loaded onto (a) SDS-PAGE; lane 1, crude cell lysate; lane 2, purified E. coli expressed bovine brain NMT2. (b) Purified E. coli expressed bovine brain NMT2 (thirty microgram) immunoblotted with monoclonal anti-NMT2 (1 : 1000 dilution) as described in Section 2.

Mentions: Subsequently, the cDNA of NMT2 was subcloned in to the expression vector pQE9 and transformed in to E. coli M13 (pREP4). For the purification of recombinant brain NMT2, the crude cell lysate was applied to Ni-NTA Agarose column and the bound His6-NMT2 was eluted as described in the experimental procedures. This single step purification was sufficient to produce highly purified recombinant brain NMT2 as judged by coomassie staining of samples resolved by SDS-PAGE (Figure 3(a)). The molecular mass of purified brain NMT2 was 50 kDa. Furthermore, a monoclonal antibody raised against brain NMT2 was immunoreactive towards the recombinant brain NMT2 protein (Figure 3(b)). This is in general agreement with previous studies of other NMTs that gave molecular mass of 50–60 kDa for monomeric human [29, 30], 50 kDa for bovine spleen [31] and cardiac muscle [20], 55 kDa for yeast [32], 53 kDa for Candida albicans [33], and 46 kDa for Drosophila [34]. However, NMTs from murine leukemia cell line L1210 [35] and bovine brain [13] have been demonstrated to exist in multiple isoforms.


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

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

SDS-PAGE and Western blot analysis of bovine brain NMT2. Thirty microgram of proteins was loaded onto (a) SDS-PAGE; lane 1, crude cell lysate; lane 2, purified E. coli  expressed bovine brain NMT2. (b) Purified E. coli  expressed bovine brain NMT2 (thirty microgram) immunoblotted with monoclonal anti-NMT2 (1 : 1000 dilution) as described in Section 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: SDS-PAGE and Western blot analysis of bovine brain NMT2. Thirty microgram of proteins was loaded onto (a) SDS-PAGE; lane 1, crude cell lysate; lane 2, purified E. coli expressed bovine brain NMT2. (b) Purified E. coli expressed bovine brain NMT2 (thirty microgram) immunoblotted with monoclonal anti-NMT2 (1 : 1000 dilution) as described in Section 2.
Mentions: Subsequently, the cDNA of NMT2 was subcloned in to the expression vector pQE9 and transformed in to E. coli M13 (pREP4). For the purification of recombinant brain NMT2, the crude cell lysate was applied to Ni-NTA Agarose column and the bound His6-NMT2 was eluted as described in the experimental procedures. This single step purification was sufficient to produce highly purified recombinant brain NMT2 as judged by coomassie staining of samples resolved by SDS-PAGE (Figure 3(a)). The molecular mass of purified brain NMT2 was 50 kDa. Furthermore, a monoclonal antibody raised against brain NMT2 was immunoreactive towards the recombinant brain NMT2 protein (Figure 3(b)). This is in general agreement with previous studies of other NMTs that gave molecular mass of 50–60 kDa for monomeric human [29, 30], 50 kDa for bovine spleen [31] and cardiac muscle [20], 55 kDa for yeast [32], 53 kDa for Candida albicans [33], and 46 kDa for Drosophila [34]. However, NMTs from murine leukemia cell line L1210 [35] and bovine brain [13] have been demonstrated to exist in multiple isoforms.

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