Location of age-related modifications in rat muscle glyceraldehyde-3-phosphate dehydrogenase.

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.