The kinetic locking-on strategy for bioaffinity purification: further studies with bovine liver glutamate dehydrogenase.
The locking-on strategy uses soluble analogues of the enzymes specific substrate to produce biospecific adsorption of individual NAD(P)(+)-dependent dehydrogenases on immobilized NAD(P)(+) derivatives, which is so selective that a single enzyme activity can be purified from crude cellular extracts in a single chromatographic step with yields approaching 100%. However, attempts to further develop and apply this strategy to the biospecific chromatographic purification of a range of NAD(P)(+)-dependent dehydrogenases revealed some anomalous chromatographic behavior and certain unexplained phenomenon. Much of this can be attributed to nonbiospecific interference effects. Identification and elimination of this interference is discussed in the present study focusing on bovine liver glutamate dehydrogenase (GDH; EC 1.4.1.3) as the "test" enzyme. Results further confirm the potential of the locking-on strategy for the rapid purification of NAD(P)(+)-dependent dehydrogenases and provide further insight into the parameters which should be considered during the development of a truly biospecific affinity chromatographic system based on the locking-on strategy. The kinetic mechanism of bovine liver GDH has been the topic of much controversy with some reports advocating a sequential ordered mechanism of substrate binding and others reporting a sequential random mechanism. Since the kinetic locking-on strategy is dependent on the target NAD(P)(+)-dependent dehydrogenase having an ordered sequential mechanism of substrate binding, the bioaffinity chromatographic behavior of bovine liver GDH using the locking-on tactic suggests that this enzyme has an ordered sequential mechanism of substrate binding under a variety of experimental conditions when NAD(+) is used as cofactor.
O'Flaherty
M
M
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
McMahon
M
M
Forde
J
J
Mulcahy
P
P
D000494:Allosteric Regulation; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013379:Substrate Specificity
D008024:Ligands; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase