Affinity Electrophoresis Used for Analses on the Interactions Between Dehydrogenases and Their Cofactors

Analyses on the interactions between dehydrogenases and their cofactors by utilizing affinity electrophoresis (AEP) technique are described. Dissociation constants of NAD-dependent dehydrogenases (NAD-DH) and NADP-dependent dehydrogenases (NADP-DH) for cofactor analogues, 5'-AMP and 2'AMP, were determined by AEP. A comparison of those dissociation constants suggests :(a) NAD-DH and NADP-DH from different sources have diverse affinities to 5'-AMP and 2'-AMP, (b) a phosphate group at position of 2' in ribose moiety of 2'-AMP and NADP play an important role in the cofactor binding onto NADP-DH, (c) lactate dehydrogenase (LDH) isozymes which have more M subunits (skeletale muscle type subunit) in their tetrameric structure have the stronger affinity to 5'AMP. From these observations, it will be concluded that AEP is useful not only for separation of enzymes according to their biological activities but also for analysis of the effect of subunits interaction on the cofactor binging to enzymes which have different types of subunit in oligomeric structure.