title: Isatin binding proteins in rat brain: in situ imaging, quantitative characterization of specific [3H]isatin binding, and proteomic profiling.
abstract: Isatin (indole-2,3-dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [(3)H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real-time beta-imager, [(3)H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5-aminocaproyl-isatin-Sepharose, we have investigated the distribution of [(3)H]isatin specific binding sites in the rat brain, characterized their K(d) and B(max), and identified some individual brain isatin binding proteins. The binding of [(3)H]isatin to rat brain sections was saturable and characterized by K(d) values (of 0.2-0.3 microM) consistent with physiological concentrations. The highest B(max) was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [(3)H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X-100 lysates of whole-brain homogenates on 5-aminocaproyl-isatin-Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde-3-phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton-related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin.
authors.affiliation: INSERM UMRS 952, CNRS UMR 7224, UPMC Paris VI, Paris, France.
authors.forename: Michele
authors.initials: M
authors.lastname: Crumeyrolle-Arias
authors.forename: Olga
authors.initials: O
authors.lastname: Buneeva
authors.forename: Victor
authors.initials: V
authors.lastname: Zgoda
authors.forename: Arthur
authors.initials: A
authors.lastname: Kopylov
authors.forename: Ana
authors.initials: A
authors.lastname: Cardona
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Tournaire
authors.forename: Vladimir
authors.initials: V
authors.lastname: Pozdnev
authors.forename: Vivette
authors.initials: V
authors.lastname: Glover
authors.forename: Alexei
authors.initials: A
authors.lastname: Medvedev
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D001923:Brain Chemistry; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism; D007031:Hypothalamus / Q000033:anatomy & histology / Q000378:metabolism; D007510:Isatin / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D014316:Tritium / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; D014316:Tritium; D007510:Isatin; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)