The use of auramine O to study ligand binding and subunit cooperativity of lactate dehydrogenase.
The tetrameric molecule of pig skeletal muscle lactate dehydrogenase binds a cationic fluorescent probe, auramine O, at four equal non-interacting sites with a dissociation constant of (1.25 +/- 0.2) X 10(-4) M. Fluorescence of the dye/enzyme mixture is strongly pH-dependent, with a maximum at pH 6.3-6.8. Auramine O-binding sites are located outside the active center of the enzyme. The microenvironment of the bound dye changes upon interaction of lactate dehydrogenase with NAD+, NADH, ADP and pyruvate. The binding of specific ligands induces an increase in fluorescence of auramine O-enzyme complex. This effect was used to determine the dissociation constants of the complexes of lactate dehydrogenase with specific ligands. Pyruvate was demonstrated to bind to the apoenzyme-auramine O complex with a dissociation constant of 5.2 X 10(-4) M. With the use of auramine O, it became possible to reveal subunit interactions within the tetrameric molecule of lactate dehydrogenase. They are manifested in the changes of the microenvironment of a dye-binding site located on one of the subunits induced by the binding of ligands in the active center of a neighboring subunit.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D001576:Benzophenoneidum / Q000378:metabolism*; D005470:Fluorometry; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D046911:Macromolecular Substances; D008433:Mathematics; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013552:Swine
D000814:Aniline Compounds; D046911:Macromolecular Substances; D011773:Pyruvates; D009243:NAD; D001576:Benzophenoneidum; D000244:Adenosine Diphosphate; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase