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Zusammenfassung
Crosslinking with glutaraldehyde was applied to the study of the assembly of lactate dehydrogenase from pig heart. To eliminate perturbations caused by excessive folding reactions, acid dissocn. was performed in the presence of 0.8M Na2SO4 at 0 Deg. Under optimum conditions complete crosslinking of the tetrameric enzyme was achieved in <2 min. Crosslinking during reconstitution proves the dimer ...
Zusammenfassung
Crosslinking with glutaraldehyde was applied to the study of the assembly of lactate dehydrogenase from pig heart. To eliminate perturbations caused by excessive folding reactions, acid dissocn. was performed in the presence of 0.8M Na2SO4 at 0 Deg. Under optimum conditions complete crosslinking of the tetrameric enzyme was achieved in <2 min. Crosslinking during reconstitution proves the dimer to be the only intermediate of reassocn.. The dimer-tetramer transition is rate-limiting for both reassocn. and reactivation, suggesting the tetramer to be the enzymically active species. The presence of monomers during reconstitution indicates that tetramer formation is preceded by a fast monomer-dimer equil. The kinetic model describing the exptl. data is: 4 monomers .dblharw. 2 dimers (with equil. const. K) and 2 dimers -> 1 tetramer (with a 2nd-order rate const. k). K Was 3 * 107 L mol-1, and k was 1.4 * 104 L mol-1.
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