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Simultaneous determination of the reduction of NAD + and NADP + by glutamate dehydrogenase supports preferential utilization of NAD + in glutamate oxidation
Author(s) -
Treberg Jason Robert,
Brosnan Margaret E.,
Brosnan John T.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a664-d
Subject(s) - nad+ kinase , cofactor , glutamate dehydrogenase , glycerol 3 phosphate dehydrogenase , dehydrogenase , allosteric regulation , chemistry , biochemistry , stereochemistry , enzyme , glutamate receptor , receptor
The mitochondrial enzyme glutamate dehydrogenase ([GDH] E.C. 1.4.1.3 ) is unusual amongst vertebrate NAD(P) + dependent dehydrogenases in that it can utilize either NAD + or NADP + , and their reduced counterparts, with similar efficiency. Although NADP + typically displays lower maximal velocities, the apparent affinity of GDH for this cofactor is somewhat lower than that for NAD + . The goal of the present study was to determine which cofactor is preferentially reduced when both are present at equal concentrations. First we describe methodology developed for the simultaneous determination of NAD + and NADP + reduction by GDH. We then apply this technique to determine which cofactor, NAD + or NADP + , is preferentially reduced when present in equimolar concentrations, particularly in the presence of known allosteric effectors of GDH, including inhibitory (GTP) as well as activating (leucine and ADP) compounds and combinations thereof. The reduction of NADP + is unaffected by equimolar amounts of NAD + whereas the rate of NAD + reduction is inhibited by the presence of NADP + . Moreover, NADP + shows a modestly higher relative activation by ADP and leucine. However, under all conditions tested, the absolute rate of NAD + reduction was approximately 2–2.5 times that of NADP + , indicating that when both cofactors are present, NAD + is preferentially reduced relative to the rate of NADP + reduction.