title: Purification of glutamate dehydrogenase from liver and brain.
abstract: Two alternative procedures are described for the purification of the major form of glutamate dehydrogenase (L-glutamate-NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3: GDH) from ox liver and brain. The first involves affinity chromatography on a column of the allosteric inhibitor GTP bound to Sepharose, whereas the other uses a bifunctional ligand (bis-NAD+) composed of two NAD+ molecules linked together by a spacer arm to precipitate the enzyme in the presence of the substrate analogue glutarate. In both procedures the affinity steps are preceded by ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose. Procedures for the synthesis of GTP-Sepharose and bis-NAD+ are described and the ancillary procedures, including the assay of GDH activity and the determination of protein concentration, are also presented.
authors.affiliation: Department of Biochemistry Trinity College, Dublin, Ireland.
authors.forename: Martha
authors.initials: M
authors.lastname: Motherway
authors.affiliation: Department of Biochemistry Trinity College, Dublin, Ireland.
authors.forename: Keith F
authors.initials: KF
authors.lastname: Tipton
authors.affiliation: GlaxoSmithKline, Middlesex, United Kingdom.
authors.forename: Alun D
authors.initials: AD
authors.lastname: McCarthy
authors.affiliation: Centre National de la Recherche Scientifique, Rennes, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Couée
authors.affiliation: University College Dublin, Dublin, Ireland.
authors.forename: Jane
authors.initials: J
authors.lastname: Irwin
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry / Q000378:metabolism
chemical_list: D009243:NAD; D006160:Guanosine Triphosphate; D012685:Sepharose; D005969:Glutamate Dehydrogenase