Lactate dehydrogenase in Phycomyces blakesleeanus

Abstract

1. An NAD-specific L(+)-lactate dehydrogenase (EC 1.1.1.27) from the mycelium of Phycomyces blakesleeanus N.R.R.L. 1555 (-) was purified approximately 700-fold. The enzyme has a molecular weight of 135,000-140,000. The purified enzyme gave a single, catalytically active, protein band after polyacrylamide-gel electrophoresis. It shows optimum activity between pH 6.7 and 7.5. 2. The Phycomyces blakesleeanus lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate, and its coenzyme, NADH, at pH 7.5, indicating the existence of multiple binding sites in the enzyme for these ligands. 3. At pH 6.0, the enzyme shows high substrate inhibition by pyruvate. 3-hydroxypyruvate and 2-oxovalerate exhibit an analogous effect, whereas glyoxylate does not, when tested as substrates at the same pH. 4. At pH 7.5, ATP, which inhibits the enzyme, acts competitively with NADH and pyruvate, whereas at pH 6.0 and low concentrations of ATP it behaves in a allosteric manner as inhibitor with respect to NADH, GTP, however, has no effect under the same experimental conditions. 5. Partially purified enzyme from sporangiophores behaves in entirely similar kinetic manner as the one exhibited by the enzyme from mycelium.

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

“…Since about half of adenylate kinase is in the mitochondrial intermembrane space, these results indicate that cytosolic LDH and cytosolic (non mitochondrial) AK are released at the same digitonin concentration. At the next lower concentration of digitonin (4 μM), relatively more of the cytosolic AK pool was released than LDH, consistent with the smaller molecular weight of cytosolic AK (21.7 kDa) [29] compared to LDH (140 kDa) [30]. The second pool of AK (∼45% of activity) was released by higher digitonin, and essentially all AK activity was released by 80 μM digitonin (Fig.…”

“…Mitochondrial metabolic alterations after ethanol exposure include increased respiration and acetaldehyde oxidation, decreased fatty acid oxidation and ATP generation, glutathione depletion, and oxidative stress [1–8,29,30]. Normal mitochondrial metabolism requires continuous exchange of metabolites between the cytosol and the mitochondrial matrix.…”

Glycerol Dehydrogenase Plays a Dual Role in Glycerol Metabolism and 2,3-Butanediol Formation in Klebsiella pneumoniae

“…The enzyme activities of both DhaD and GldA increase at higher NADH concentrations, possibly suggesting a positive cooperativity between GDHs and NADH. Similar phenomena have been observed in a previously reported NADH/NAD ϩ -dependent dehydrogenase, and a Hill plot with a Hill coefficient was used to verify this cooperativity (36). Hill plots of the data obtained from plots of enzyme activity (v) versus concentration of NADH produced lines with maximal slopes (h value) greater than or close to 2 (2.32 for DhaD and 1.77 for GldA) (Fig.…”

“…To perform Hill plots analyses, 100 mM (3R)/(3S)-AC and modified NADH concentrations (0.025-0.5 mM) were used. The Hill coefficient (h) was obtained from the Hill plots (36).…”

Functional Characteristics of Pyruvate Transport inPhycomyces blakesleeanus

“…We cannot exclude the possibility that some fermentation products, such as lactate, may withdraw with H ϩ to the outside of Phycomyces mycelium and contribute to the acidification. Notably, lactate dehydrogenase in Phycomyces is a regulatory enzyme, controlled by the energetic charge (Soler et al, 1982;Busto et al, 1983). On the other hand, when glucose and pyruvate were added simultaneously, D-glucose behaved as a potent noncompetitive inhibitor of pyruvate transport in P. blakesleeanus at pH 6.0.…”