J. Biol. Chem., Vol. 256, Issue 17, 8875-8877, Sep, 1981
Location of age-related modifications in rat muscle glyceraldehyde-3- phosphate dehydrogenase
A Gafni
The interactions of rat muscle glyceraldehyde-3-phosphate dehydrogenase
purified from young and old animals with NADH and with the fluorescent
analogue nicotinamide 1,N6-ethenoadenine dinucleotide were investigated.
While the spectra of the circular polarization of fluorescence emitted by
the ethenoadenine derivative when bound to the two enzyme preparations were
identical large differences were revealed between the corresponding spectra
in the case of NADH. From these results it was concluded that age-related
modifications occur in the nicotinamide binding sites, but not in the
adenine binding sites of this enzyme. The circular polarization of
fluorescence of the ethenoadenine derivative was found to depend on the
stoichiometry of its complexes with the enzyme while the spectra obtained
for NADH were independent of the degree of saturation of the coenzyme
binding sites. These observations demonstrate that progressive structural
changes occur at the adenine site as a function of coenzyme saturation.
These changes may be responsible for the strong negative cooperative in
coenzyme binding. The finding that only the nicotinamide binding sites are
affected by age explains our previous observation that while the affinity
toward coenzyme binding which depends on both adenine and nicotinamide
moieties is reduced upon aging the negative cooperativity of binding is not
significantly changed, since this latter property depends on the state of
the adenine site only.
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