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NADH binding to porcine mitochondrial malate dehydrogenase
The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were ob...
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Published in: | The Journal of biological chemistry 1979-09, Vol.254 (18), p.9059-9062 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium
and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence
of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate,
measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant
for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence
of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant
for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation
from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high
as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to
demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration
on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(19)86808-4 |