Further studies on the bioaffinity chromatography of NAD(+)-dependent dehydrogenases using the locking-on effect.
Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described using the tactic.
O'Carra
P
P
Department of Biochemistry, University College, Galway, Ireland.
Griffin
T
T
O'Flaherty
M
M
Kelly
N
N
Mulcahy
P
P
D000345:Affinity Labels; D000818:Animals; D001391:Azo Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D008291:Malate Dehydrogenase / Q000302:isolation & purification; D015394:Molecular Structure; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D010070:Oxalates / Q000494:pharmacology; D010088:Oxidoreductases / Q000302:isolation & purification*; D013552:Swine; D015003:Yeasts
D000345:Affinity Labels; D001391:Azo Compounds; D004791:Enzyme Inhibitors; D010070:Oxalates; D009243:NAD; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; D005969:Glutamate Dehydrogenase