Reassociation of Lactic Dehydrogenase from Pig Heart Studied by Cross-Linking with Glutaraldehyde

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Abstract
Cross-linking with glutaraldehyde has been successfully applied in order to analyze the kinetics of reassociation of oligomeric enzymes (R. Hermann, R. Rudolph, and R. Jaenicke Nature 277, 243-245 (1979)). In the present study the assembly of lactic dehydrogenase from pig heart is investigated using this approach. In order to eliminate perturbations caused by excessive folding reactions, acid dissociation was performed in the presence of 0.8 M Na2SO4 at 0°C . Under optimum conditions complete cross-linking of the tetrameric enzyme was achieved in less than 2 minutes. Cross-linking during reconstitution proves the dimer to be the only intermediate of reasso­ ciation. The dimer → tetramer transition is found to be rate-limiting for both reassociation and reactivation, suggesting the tetramer to be the enzymatically active species. The presence of monomers during reconstitution indicates that tetramer formation is preceded by a fast monomer-dimer equilibrium. The kinetic model describing the experimental data is characterized by an equilibrium constant K = 3 ± 1 · 107 liter · m ol-1, and a second-order rate constant k = 1.4 ± 0.2 · 104 liter · mol-1 · s-1.