Preferential recognition of undisruptable dimers of inducible nitric oxide synthase by a monoclonal antibody directed against an N-terminal epitope.
Overproduction of NO by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer in which the subunits align in a head-to-head manner, with the N-terminal oxygenase domains forming the dimer interface and a zinc metal center stabilizing the dimer. Thus, dimerization represents a critical locus for therapeutic interventions for regulation of NO synthesis. We have recently shown that intracellular iNOS forms dimers that are "undisruptable (UD)" by heat, SDS, strong denaturants, and/or reducing agents. Our data further suggest that the zinc metal center plays a role in forming and/or stabilizing iNOS undisruptable dimers (UD-dimers). In this study, we show that a mAb directed against a unique epitope at the oxygenase domain of human iNOS preferentially recognizes UD-dimers. This observation has implications for the mechanism of formation and regulation of dimer formation of iNOS. Our data suggest that UD-dimers of iNOS, in spite of SDS-PAGE denaturation, still maintain features of the quaternary structure of iNOS particularly at its N-terminal end and including head-to-head contact of the oxygenase domains.
Mazumdar
Tuhina
T
Pulmonary and Critical Care Section, Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Eissa
N Tony
NT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002460:Cell Line; D016384:Consensus Sequence; D019281:Dimerization; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011489:Protein Denaturation; D013045:Species Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse