The Membrane-bound L- and D-lactate dehydrogenase activities in mitochondria from Euglena gracilis.
The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for L- and D-lactate were similar, but the V(max) was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q(1), reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both L- and D-lactate. Oxalate exerted a mixed-type inhibition regarding L- or D-lactate and also against DCPIP. The rate of L-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of L-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the D-iLDH for both inhibitors, D-iLDH thermo-stability at 70 degrees C and denaturation of L-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass.
Jasso-Chávez
R
R
Departamento de Bioquímica, Facultad de Medicina, UNAM, México, Mexico.
Torres-Márquez
M E
ME
Moreno-Sánchez
R
R
D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D004795:Enzyme Stability; D005056:Euglena gracilis / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D045022:Lactate Dehydrogenases; D019344:Lactic Acid / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D012995:Solubility; D013237:Stereoisomerism
D008565:Membrane Proteins; D019344:Lactic Acid; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)