Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate dehydrogenase from Actinomyces viscosus.
A nicotinamide adenine dinucleotide-specific L-(+)-lactate dehydrogenase (LDH) (EC 1.11.27) from Actinomyces viscosus T-6-1600 was purified approximately 110-fold by a combination of diethylaminoethyl-cellulose and 0.5 M Agarose A column chromatography. The ldh was stable at 26 C, but was quite labile at temperatures below 5 C. The enzyme had a molecular weight of 100,000 +/- 10,000 as determined by 0.5 M Agarose molecular exclusion chromatography and showed optimum activity between pH 5.5 and 6.2. The A. viscosus LDH exhibited homotropic interactions with its substrate, pyruvate, and its coenzyme, reduced nicotinamide adenine dinucleotide, indicating multiple binding sites on the enzyme for these ligands with some degree of cooperative interaction between them. The enzyme was under negative control by adenosine 5'-triphosphate, and its kinetic response to the negative effector was sigmoidal in nature. Inorganic phosphate reversed the inhibition exerted on the A. viscosus LDH by adenosine. The 5'-triphosphate thermal stability at 65 C of the LDH from A. viscosus was increased in the presence of its negative effector, adenosine 5'-triphosphate, but was markedly decreased in the presence of its coenzyme, reduced nicotinamide adenine dinucleotide. The glycolytic intermediate, fructose-1,6-diphosphate, had no effect on the catalytic activity of the A. viscosus LDH at saturating pyruvate concentrations. However, fructose-1,6-diphosphate was a potent positive effector at low substrate concentrations. Thus the A. viscosus LDH is under positive control by fructose-1,6-diphosphate and inorganic phosphate, but under negative control by adenosine 5'-triphosphate.
D000190:Actinomyces / Q000201:enzymology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002474:Cell-Free System; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D005636:Fructosephosphates / Q000494:pharmacology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism; D013696:Temperature
D005636:Fructosephosphates; D007773:Lactates; D010710:Phosphates; D011773:Pyruvates; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase