Abstract
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.
Highlights
Others to measure the enzymatic activity of monomer-mitochondrial malate dehydrogenase have utilized extremes of pH or denaturing conditions [10]and have been unsuccessful
The specific activity a t each gel containing immobilized mitochondrial malate dehydrogenase as pH wascalculatedbased on protein quantification of immobilized described below
In a study of transaldolase, activation of Sepharose gel with 5 mg of CNBr/ml of packed gel produced a sufficiently low level of activation to allow linkage of the tetrameric protein through a single subunit [25].In a recent studyof immobilized alkaline phosphatase, the CNBr to resin ratio had to be reduced to 1.0 m g / d of packed gel or less to achieve a sufficiently low density of linkage sites [20].In that studyg, el activation was allowed to proceed for 12 min at room temperatureandactivationwith 0.5-1.0 mg of CNBr/ml of gel resulted in 100-200 pg of proteinbound/ml of gel
Summary
Assays in the NADH + mitochondrial malate dehydrogenase/ml of packed gel. Assays of immobilized mitochondrial malate dehydrogenase in the NAD’ NADH direc- The specific activity a t each gel containing immobilized mitochondrial malate dehydrogenase as pH wascalculatedbased on protein quantification of immobilized described below.
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