The effect of divalent metal cations on the functioning of peroxisomal malate dehydrogenase from maize leaves
The enzymes of the Krebs cycle in the plant cell are usually represented by several isoforms localized in the mitochondrial, peroxisomal and other plant cell fractions. The physiological role of isoenzymes of malate dehydrogenase is now well studied for the mitochondrial form of this enzyme. The aim of this work was to study the effect of divalent metal ions on the activity of peroxisomal MDH in the mesophyll of maize. Modified multistage purification of the peroxisomal isoform malate dehydrogenase (E.C. 1.1.1.37) from the maize maize mesophyll, including isotopic centrifugation with a step gradient of sucrose at a concentration of 1.3 to 2.5 M. The separation of subcellular fractions (peroxisomes, mitochondria and cytosol) was performed by ultracentrifugation at 100,000 g. The determining value for effective purification of the enzyme was ion-exchange chromatography, which made it possible to obtain homogeneous preparations of malate dehydrogenase from the maize mesophyll of maize leaves. The degree of purification of the enzyme, which has a specific activity of 513 U / mg protein, was 135 times, and the yield was 4%. The use of ionexchange chromatography with the use of DEAE-Sephacel for ion-detection in the quality opens up prospects for its use for fine separation of molecular isoforms of oxidative enzymes close to tricarboxylic acid cycles. The preparation of the peroxisomal form of the enzyme preparations studied in the electrophoretical-ly homogeneous state made it possible to conduct a comprehensive study of the regulatory aspects of the functioning of MDH. On the obtained preparation, the influence of divalent cations such as calcium, barium, magnesium and manganese was investigated. The type and nature of the regulating effect of the cations under study on the malate dehydrogenase system were determined by the graphoanalytical method. It is shown that magnesium ions in small doses (up to 2 mM) exert an activating effect on the enzyme activity, and in large (more than 2 mM) - inhibit the activity of malate dehydrogenase. Other cations studied had an inhibitory effect on the functioning of the enzyme. Thus, an analysis of the data obtained indicates that the divalent cations studied can participate in the regulation of the catalytic action of the malate dehydrogenase enzyme system in the mesophyll of Zea mays leaves.