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 g racilis, 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 d isplayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q(1), rea cting as the second substrate with the modified, reduced enzyme. Oxamate wa s 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 tha n the rate of dehydrogenation, which was mersalyl-insensitive, These data s uggested that the active site of L-iLDH was orientated toward the intermemb rane space. The following observations indicated the existence of two stere o-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a gr eater affinity of the D-iLDH for both inhibitors, D-iLDH thermo-stability a t 70 degreesC and denaturation of L-iLDH, opposite signs in the enthalpy ch ange for the association reaction of the isomers to the enzyme, differentia l solubilization of both activities with detergents, and different molecula r mass. (C) 2001 Academic Press.