Reversal of age-related effects in rat muscle phosphoglycerate kinase.
AUTOR(ES)
Yuh, K C
RESUMO
Rat muscle phosphoglycerate kinase is one of several enzymes in which age-related effects have been identified. Thus, samples of this enzyme isolated from old rats display a greatly increased heat stability as compared with enzyme isolated from young animals. Previous studies detected no differences in the sequence of amino acids or in the net charge between the young and old forms of the enzyme and it was concluded that the age-related structural modifications are purely conformational. The present study was conducted with the aim of critically testing this hypothesis. To this end, samples of phosphoglycerate kinase purified from skeletal muscle of young and old rats were unfolded by an 18-hr incubation in a 2 M guanidine hydrochloride solution at 4 degrees C, a treatment that results in extensive loss of the three-dimensional structure of the enzyme. A complete reactivation of both enzymes was achieved by dilution of the unfolded enzyme solutions into a large excess of denaturant-free buffer followed by 4 hr of incubation at 25 degrees C. The reactivation kinetics of the unfolded young and old enzymes were practically identical and the refolded products, compared using heat-inactivation kinetics as a sensitive probe, were found to be identical. Moreover, their heat inactivation coincided with that of young untreated phosphoglycerate kinase. These results demonstrate the reversibility of age-related effects at the molecular level and provide strong support for the hypothesis that the modifications in phosphoglycerate kinase in old muscle are purely conformational and, hence, clearly postsynthetic.
