ABSTRACT
A simple method was developed to determine histamine, an important compound in chemical food poisoning, by extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry using a hydrophilic column with sulfobetaine-type zwitterion groups. The quantitation range in seafood products was from 0.4 to 200 mg kg(-1) for 5 g food samples. Quantitative recoveries were obtained with four types of seafood product. These results agreed well with those from the more complex, conventional HPLC method, which requires sample derivatization with dansyl chloride.
Subject(s)
Chromatography/methods , Food Analysis/methods , Histamine/analysis , Hydrophobic and Hydrophilic Interactions , Seafood/analysis , Tandem Mass Spectrometry/methods , Histamine/chemistry , Spectrometry, FluorescenceABSTRACT
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.
Subject(s)
Acyltransferases/metabolism , Antibodies, Monoclonal/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Acyltransferases/antagonists & inhibitors , Acyltransferases/immunology , Amino Acid Sequence , Antibody Specificity , Biotin/metabolism , Chromatography, High Pressure Liquid/methods , Glycine/analogs & derivatives , Glycine/analysis , Glycine/immunology , HIV-1/genetics , HIV-1/immunology , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/immunology , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Streptavidin/chemistryABSTRACT
N-myristoyltransferase (NMT) is essential for the survival of eukaryotes and the production of infectious human immunodeficiency virus type-1(HIV-1) by the host cell. In this study, we found decreases in the mRNA levels of human NMT isoforms and the NMT activities in the course of HIV-1 infection in the human T-cell line, CEM. Investigating the cytotoxic effect of the novel synthetic NMT inhibitors on the chronic HIV-1 infected T-cell line, CEM/LAV-1, and the uninfected CEM, revealed that the cytotoxic effect was significantly selective for CEM/LAV-1. This was thought to be due to the difference between the NMT levels of the cell lines. In this paper, we propose that NMT may be a candidate target for anti-HIV-1-infected-cell agents.
