Factors affecting the quaternary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei and Lactobacillus curvatus as investigated by hybridization and ultracentrifugation.

The allosteric L-lactate dehydrogenases of Lactobacillus curvatus and Lactobacillus casei exist in the tetrameric from (molecular weight about 145 000) at pH 5.0--5.5 even in the absence of the effectors Mn2+ and Fru(1,6)P2 (fructose 1,6-bisphosphate), but undergo reversible dissociation to monomers (molecular weight about 35 000) at higher pH values or in the presence of urea. In the range between pH 5.5 (tetrameric state) and pH 7.4 (monomeric state) the L. curvatus L-lactate dehydrogenase exists in a dissociation-association equilibrium comprising tetramers, dimers and monomers as indicated by the Sc20,w values and the results of hybridization experiments. The simultaneous addition of both effectors [Mn2+ and Fru(1,6)P2] at pH 7.4, however, resulted in the stabilization of the tetrameric form. The addition of Fru(1,6)P2 alone at pH 7.4 had almost no influence on the quarternary structure, whereas the addition of Mn2+, as well as that of NADH, largely prevented dissociation. The L-lactate dehydrogenase of L. casei showed similar properties, although the enzyme dissociates only at about pH greater than or equal to 7.8. As in the case of the L. curvatus enzyme, Fru(1,6)P2 has no influence on the pH-dependent dissociation of the L. casei enzyme, whereas Mn2+ stabilizes the tetrameric structure. Reconstitution of a mixture of the two dissociated enzymes results in the formation of all statistically possible, enzymatically active hybrids. No hybridization between the allosteric enzymes from L. casei and L. curvatus and the non-allosteric ones of Lactobacillus plantarum and Lactobacillus acidophilus was observed.