Kinetics of reconstitution of porcein muscle lactic dehydrogenase after reversible high pressure dissociation.

Porcine muscle lactic dehydrogenase can be reversibly dissociated into monomers at high hydrostatic pressure. The rate of dissociation depends on the conditions of the solvent (Schade et al., 1980, Biochemistry, in press). Maximum yields of reactivation are achieved after dissociation by 20 min incubation in 0.2 M Tris/HCl buffer or 0.2 M KCl at pH 7.6, in the presence of 10 mM dithioerythritol and 1 mM EDTA, provided that both dissociation and reassociation are performed under anaerobic conditions. At enzyme concentrations of the order of 1 microM reactivation amounts to greater than or equal to 95%, the product of reactivation being indistinguishable from the enzyme in its initial native state. Based on the long-term stability of the enzyme under the optimum given conditions of reactivation, the kinetics of reconstitution after pressure release were investigated over a wide range of enzyme concentrations (1 nM less than c less than 1 microM). The weakly sigmoidal kinetics may be described by an irreversible uni-bimolecular reaction scheme, corresponding to a sequential transconformation-association process. Assuming the protomers to be enzymatically inactive, the kinetic profiles may be fitted by one set of kinetic constants: kuni=1.5 X 10(-2) s-1 and kbi=7 X 10(3) s-1 M-1, the association step belonging to either dimer or tetramer formation.