Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A simplified assay for the enzyme responsible for the attachment of myristic acid to the N-terminal glycine residue of proteins, myristoyl-CoA: glycylpeptide N-myristoyltransferase.

ABSTRACT: A greatly simplified assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase (NMT) activity is described. The assay is based on the differential solubility of the acyl-peptides produced as a consequence of the NMT activity and yields results comparable with those obtained with the original assay described by Towler & Glaser [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2812-2816], which requires h.p.l.c. to determine the production of the acyl-peptides. The use of the revised assay in the preliminary steps of the purification of rat brain NMT is described, and its use in determining the fatty acid-specificity of the enzyme is illustrated. The results are shown to be comparable with those obtained with the h.p.l.c.-based assay.

SUBMITTER: McIlhinney RA

PROVIDER: S-EPMC1133441 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Mutations of human myristoyl-CoA:protein N-myristoyltransferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae.

Project description:We have isolated cDNAs encoding human myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) by complementing the nmtl-181 mutation of Saccharomyces cerevisiae, which causes temperature-sensitive myristic acid auxotrophy. Human NMT is derived from a single-copy gene, contains 416 amino acids, is 44% identical to S. cerevisiae NMT (yeast NMT), and can complement the lethal phenotype of an nmtl null mutation. Human and yeast NMTs have overlapping yet distinct protein substrate specificities as judged by a coexpression system that reconstitutes protein N-myristoylation in Escherichia coli. Both enzymes contain a glycine five residues from the C terminus. Gly----Asp or Lys mutagenesis in these orthologous NMTs produces marked reductions in their activities in E. coli as well as temperature-sensitive myristic acid auxotrophy in S. cerevisiae. These results indicate highly conserved structure-function relationships in vivo and underscore the usefulness of these functional assays for identifying factors that regulate protein N-myristoylation in mammalian systems.

| S-EPMC525646 | biostudies-other

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

Project description: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.

| S-EPMC2737134 | biostudies-literature

Purification and partial sequencing of myristoyl-CoA:protein N-myristoyltransferase from bovine brain.

Project description:The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyses the transfer of myristic acid to the N-terminal glycine residue of cell and viral proteins. In this report the purification and partial sequencing of this enzyme from bovine brain is described. Using a combination of ammonium sulphate precipitation, chromatography on DEAE-Sepharose and affinity chromatography on CoA-agarose the enzyme was purified some 40-fold. Size-exclusion chromatography of this material in the presence of myristoyl-CoA yielded two peaks of enzyme activity with apparent molecular masses of 66 kDa and 43 kDa. Chromatography of the CoA-affinity-purified material on MONO-S followed by size-exclusion chromatography in the presence of myristoyl-CoA resulted in the isolation of the large form of the enzyme purified 3000-fold. Analysis by SDS/PAGE of this material showed a major 60 kDa silver-stained band. Similar analysis of the 43 kDa enzyme fraction from the same separation showed that this fraction contained several proteins including a major component with an apparent molecular mass of 49 kDa. Attempts at N-terminal sequencing of the 66 kDa form of the enzyme were unsuccessful and therefore this material was digested with trypsin and the resulting peptides separated by reverse-phase h.p.l.c. N-terminal protein sequencing of these peptides yielded sequences which show sequence similarity to those of yeast N-myristoyl-transferase.

| S-EPMC1132288 | biostudies-other

N-myristoylation of p60src. Identification of a myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues.

Project description:A 16-residue synthetic peptide corresponding to the N-terminal sequence of p60src was used as the acyl acceptor in an assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues. An additional C-terminal tyrosine amide was added to this peptide to facilitate radioiodination and enhance detectability. Reverse-phase h.p.l.c. enabled the simultaneous detection and quantification of the peptide substrate and its N-myristoylated product. N-Myristoyltransferase activity was highest in the brain with decreasing activities in lung, small intestine, kidney, heart, skeletal muscle and liver. Brain activity was distributed approximately equally between the 100,000 g pellet and supernatant fractions. The soluble enzyme exhibited a Kappm of 20 microM for the src peptide and an optimum between pH 7.0 and 7.5. Maximum N-acylating activity was seen with myristoyl (C14:0)-CoA with lower activities found with the C10:0-CoA and C12:0-CoA homologues. No activity was obtained with palmitoyl (C18:0)-CoA but this derivative inhibited N-myristoyltransferase activity greater than 50% at equimolar concentrations with myristoyl-CoA. With a decapeptide corresponding to the N-terminal sequence of the cyclic AMP-dependent protein kinase catalytic subunit as the acyl acceptor, the brain enzyme displayed a Kapp.m of 117 microM and was about 14-fold less catalytically effective than with the p60src acyl acceptor. Transferase activity was also seen with a 16-residue peptide corresponding to the N-terminal sequence of the HIV p17gag structural protein. Inhibition studies with shorter src peptide analogues indicated an enzyme specificity for the p60src acyl acceptor beyond 9 residues.

| S-EPMC1148882 | biostudies-other

Characterization and selective inhibition of myristoyl-CoA:protein N-myristoyltransferase from Trypanosoma brucei and Leishmania major.

Project description:The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 microM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16-66 microM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.

| S-EPMC1462705 | biostudies-other

Characterization of human and rat brain myristoyl-CoA:protein N-myristoyltransferase: evidence for an alternative splice variant of the enzyme.

Project description:Using 5'-rapid amplification of cDNA ends, we have identified an extended 5'-end of mRNA coding for human myristoyl-CoA:protein N-myristoyltransferase (NMT). PCR using primers based on this new 5'-sequence and reverse primers within the currently accepted coding sequence of the enzyme resulted in the identification of a novel splice variant of NMT. In vitro translation of these cDNAs resulted in the production of proteins with apparent molecular masses of 63 kDa and 48 kDa. Immunoprecipitation of NMT from human cell lines and immunoblotting of a range of rat tissues has identified proteins with molecular masses corresponding to those derived from these cDNAs, and provided evidence that their relative abundance differs among tissues. Our results provide evidence that this enzyme exists in different forms resulting from alternative splicing of the mRNA.

| S-EPMC1219608 | biostudies-other

A fluorescence-based assay for N-myristoyltransferase activity.

Project description:N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.

| S-EPMC3863716 | biostudies-literature

Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA.

Project description:The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C(14)-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C(14)-CoA and can also bind the far more abundant palmitoyl-CoA (C(16)-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C(18:2)-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C(16)-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C(16)-CoA. These results indicate that ACBD6 can locally sequester C(16)-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C(16)-CoA.

| S-EPMC4727424 | biostudies-literature

Mapping allostery through computational glycine scanning and correlation analysis of residue-residue contacts.

Project description:Understanding allosteric mechanisms is essential for the physical control of molecular switches and downstream cellular responses. However, it is difficult to decode essential allosteric motions in a high-throughput scheme. A general two-pronged approach to performing automatic data reduction of simulation trajectories is presented here. The first step involves coarse-graining and identifying the most dynamic residue-residue contacts. The second step is performing principal component analysis of these contacts and extracting the large-scale collective motions expressed via these residue-residue contacts. We demonstrated the method using a protein complex of nuclear receptors. Using atomistic modeling and simulation, we examined the protein complex and a set of 18 glycine point mutations of residues that constitute the binding pocket of the ligand effector. The important motions that are responsible for the allostery are reported. In contrast to conventional induced-fit and lock-and-key binding mechanisms, a novel "frustrated-fit" binding mechanism of RXR for allosteric control was revealed.

| S-EPMC5001807 | biostudies-literature

A Simplified SARS-CoV-2 Pseudovirus Neutralization Assay.

Project description:COVID-19 is an ongoing pandemic caused by the highly infectious coronavirus SARS-CoV-2 that is engaging worldwide scientific research to find a timely and effective eradication strategy. Great efforts have been put into anti-COVID-19 vaccine generation in an effort to protect the world population and block SARS-CoV-2 spread. To validate the protective efficacy of the vaccination campaign and effectively control the pandemic, it is necessary to quantify the induction of neutralizing antibodies by vaccination, as they have been established to be a correlate of protection. In this work, a SARS-CoV-2 pseudovirus neutralization assay, based on a replication-incompetent lentivirus expressing an adapted form of CoV-2 S protein and an ACE2/TMPRSS2 stably expressing cell line, has been minimized in terms of protocol steps without loss of accuracy. The goal of the present simplified neutralization system is to improve SARS-CoV-2 vaccination campaign by means of an easy and accessible approach to be performed in any medical laboratory, maintaining the sensitivity and quantitative reliability of classical serum neutralization assays. Further, this assay can be easily adapted to different coronavirus variants by simply modifying the pseudotyping vector.

| S-EPMC8071407 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data