title: Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases.
abstract: This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract. Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.
authors.affiliation: Department of Applied Biology and Chemistry, Institute of Technology, Carlow, Ireland.
authors.forename: Mary
authors.initials: M
authors.lastname: McMahon
authors.forename: Julie
authors.initials: J
authors.lastname: Tynan
authors.forename: Patricia
authors.initials: P
authors.lastname: Mulcahy
mesh_terms: D000818:Animals; D002175:Candida / Q000737:chemistry / Q000201:enzymology; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D009249:NADP / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity
chemical_list: D004800:Enzymes, Immobilized; D009249:NADP; D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+)