Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase.
Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.
Takamune
Nobutoki
N
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan.
Hamada
Hirotoshi
H
Sugawara
Hideki
H
Misumi
Shogo
S
Shoji
Shozo
S
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D001710:Biotin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives / Q000032:analysis / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D019809:Streptavidin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D009842:Oligopeptides; D011994:Recombinant Proteins; D001710:Biotin; D019809:Streptavidin; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D005998:Glycine