JBC, Vol. 251, Issue 20, 6304-6307, Oct, 1976
Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate
D. M. Bleile, J. L. Jameson and J. H. Harrison
Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a
bifunctional reagent during the inactivation of porcine heart cytoplasmic
malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The
biphasic kinetics and X-azolidine-like structure formed were similar to
those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo,
T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715).
In the cytoplasmic enzyme, however, irreversible inactivation representing
X-azolidine formation was found to be the dominant characteristic of the
interaction with pyridoxal-5'-P. Spectral evidence indicated that at total
inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or
one pyridoxal-5'-P per enzymatic active site. The presence of NADH
protected the enzyme from inactivation suggesting interaction of
pyridoxal-5'-P at or near the enzymatic active centers of this enzyme.
Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme
failed to bind NADH or at least failed to bind NADH in the same fashion as
native enzyme.
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