Age-related effects in coenzyme binding patterns of rat muscle glyceraldehyde-3-phosphate dehydrogenase

The binding of NAD+ and of its fluorescent analogue, nicotinamide 1,N6-ethenoadenine dinucleotide, to glyceraldehyde-3-phosphate dehydrogenase purified from the muscles of young and old rats was studied in detail. Binding of the natural coenzyme was followed both by spectrophotometric titration of the extrinsic absorption band of the enzyme-NAD+ complex and from the degree of quenching of fluorescence of the protein. Binding of the coenzyme analogue was monitored by using the large enhancement in its fluorescence upon forming the complex with the enzyme. Both dinucleotides showed strong negative cooperativity in binding to the enzyme, similar to that displayed in their association with the rabbit muscle enzyme. The enzyme purified from old rats displayed a markedly reduced affinity toward the two dinucleotides, compared with the enzyme isolated from young animals. The various dissociation constants of both dinucleotides from the enzyme from young rats were remarkably similar to the corresponding constants in the rabbit muscle enzyme. The degree of negative cooperativity (i.e., the ratio between the dissociation constants from high- and low-affinity binding sites) in the "young" and "old" enzyme forms was not very different. It was concluded from these results that while modifications in the subunits take place upon aging, the intersubunit interaction is not significantly affected. Increasing concentrations of ATP were found to cause a gradual decrease in the negative cooperativity of NAD+ binding, which completely disappeared in the presence of 5 mM ATP. The observation that all four binding sites of the old enzyme display the same affinity toward NAD+ when the negative cooperativity is removed excludes the possibility that this enzyme form is a mixture of native and inactive species. The different dissociation constants of NAD+ from young and old enzyme forms in the presence of 5 mM ATP also demonstrate the occurrence of age-related modifications in the structure of the individual subunits.