The Enzymically Active Unit of Lactic Dehydrogenase

Abstract

Lactic dehydrogenase from pig heart isoenzyme (H 4 ) and skeletal muscle (isoenzyme M 4 ), under solvent conditions comparable to conditions in vivo, represents a stable, monodisperse tetramer. Solvent conditions which favour subunit dissociation on principle affect the native conformation of the enzyme leading to (partial) deactivation. In the case of pH‐dependent dissociation, the breakdown of the quaternary structure is preceded by deactivation. The action of high electrolyte concentration depends on the ions applied to the enzyme, deactivation following the lyotropic series of ions. Dissociation (hybridization) is promoted only by weak‐structure‐breaking ions, strong‐structure breakers leading to irreversible denaturation and aggregation. Medium electrolyte concentrations cause preferential hydration rather than dissociation. The separation of dissociation from hybrid equilibration by incubating isoenzymes H 4 and M 4 together or in two separate probes, proves that high salt concentration ( e.q. 2 M LiCl) fully dissociates the isoenzymes. Both experiments provide identical patterns for all hybrids. Low salt concentration ( e.g. 0.005 M citrate) requires low enzyme concentration and increased temperature to produce subunit dissociation. The lack of hybrid formation after separate incubation, suggests that the equilibrium in this case must be shifted to the associated state. High hydrostatic pressure favours hybrid formation, while the coenzyme (NAD and NADH) or glycine inhibit dissociation as well as hybridization. At low protein concentration, under medium conditions of the enzymic assay, no indication of subunit dissociation was observed.