Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme.
Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.
D000818:Animals; D002645:Chickens; D003593:Cytoplasm / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase