The refolding of lactate dehydrogenase subunits and their assembly to the functional tetramer.
The renaturation process of different lactate dehydrogenase isozymes (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from their unfolded subunits was investigated using a number of techniques. (a) kinetics of activity regain, (b) the kinetics of fluorescence change of fluoresecence change of the protein tryptophans, (c) kinetics of regain of the fluorescence properties of a covalently attached fluorescence probe (fluorescein) and (d) the kinetics of assembly, by following the intermediate oligomeric species appearing in the assembly pathway from monomers to tetramers. The results indicate that the unfolded polypeptide is converted to the active oligomeric species by the following scheme: Denatured subunit I leads to partially refolded subunit II leads to folded subunit III leads to dimer IV leads to tetramer. Step I and step II are first-order where step II is rate limiting. The ligands NAD+ and NADH accelerate step II, thus converting step I to the rate-limiting process. The fact that partially folded lactate dehydrogenase subunits are capable of co-enzyme binding may indicate the possible role of these ligands in the assembly of lactate dehydrogenase in vivo. Steps III and IV were found to be fast. The intermediate formation of an enzyme dimer which then dimerizes to the tetrameric species is found to be the major assembly pathway. Only a small portion of the lactate dehydrogenase tetramer is formed through the intermediate formation of a trimer intermediate.
D000818:Animals; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates; D008024:Ligands; D046911:Macromolecular Substances; D009243:NAD; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011773:Pyruvates; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis; D013552:Swine
D007527:Isoenzymes; D007773:Lactates; D008024:Ligands; D046911:Macromolecular Substances; D011773:Pyruvates; D013438:Sulfhydryl Compounds; D009243:NAD; D004228:Dithionitrobenzoic Acid; D007770:L-Lactate Dehydrogenase