The immobilization of mitochondrial malate dehydrogenase on Sepharose beads and the demonstration of catalytically active subunits.
Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been immobilized by covalent attachment to CNBr-activated Sepharose 4B-Cl gel. The gel was activated with low levels of CNBr to produce a low density of linkage sites and, hence, to facilitate linkage of the enzyme through a single subunit. Matrix-bound mitochondrial malate dehydrogenase was found to possess 50-65% of the native mitochondrial malate dehydrogenase specific activity when assayed in the NAD+ leads to NADH direction but only 5-15% of the native enzyme specific activity when assayed in the NADH leads to NAD+ direction. MB-dimeric mitochondrial malate dehydrogenase was dissociated to MB-monomer by exposure to pH 5.0 buffer. The MB-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to MB-dimer. The reconstitution of Mb-monomer to MB-dimer was accomplished by adding dissociated mitochondrial malate dehydrogenase, which exists at pH 5.0, to MB-monomer and adjusting to pH 7.5. The kinetic parameters, pH activity profile, and stability toward heat denaturation for MB-mitochondrial malate dehydrogenase (monomer and dimer) were determined and compared to native mitochondrial malate dehydrogenase. MB-mitochondrial malate dehydrogenase exhibited enhanced stability and similar pH activity profiles when compared to native mitochondrial malate dehydrogenase. Immobilization of mitochondrial malate dehydrogenase altered the enzyme's kinetic parameters in such a manner as to increase the values of Km for the substrates and decrease the values of Vmax.
D000818:Animals; D003488:Cyanogen Bromide; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D012685:Sepharose; D013552:Swine
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D012685:Sepharose; D008291:Malate Dehydrogenase; D003488:Cyanogen Bromide