The application of ion-exchange chromatography for the separation of NAD+-dependent malate dehydrogenase isoforms from maize for investigation of their thermal stability and thermolability
Abstract
NAD+-malate dehydrogenase (NAD+-MDH, EC 1.1.1.37) is a common enzyme playing an important role in many metabolic processes such as TCA, nitrogen exchange and NADH transport. Different environmental conditions strongly affect the activity of enzymes, activating or inhibiting them. The aim of this study was the investigation of the changes in the activity of malate dehydrogenases under the action of various temperatures.
The object of the study was 10-day-old Zea mays seedlings grown hydroponically. Malate dehydrogenase isozymes from maize leaves were purified using a four-stage purification, which included the stages of homogenization of plant material, protein precipitation with ammonium sulphate at the saturation of 25-80 %, gel filtration using Sephadex G-25 and ion exchange chromatography on a column packed with DEAE-Sephacel. Elution was carried out with a linear gradient of sodium chloride at a concentration from 0 to 150 mM and the fractions with malate dehydrogenase activity were collected. The measurement of enzyme activity was carried out spectrophotometrically based on the determination of the formation or consumption rate of NADH at a wavelength of 340 nm. Electrophoretic studies were carried out in polyacrylamide gel followed by universal staining of proteins with silver nitrate or a specific staining for enzymatic activity using tetrazolium method with the induction of the formation of insoluble diformazan at the site of the enzyme.
The cytoplasmic MDH preparation had a specific activity of 256 U/mg protein and a purification degree of 115. The yield was 3.5%. The specific activity of the mitochondrial form of the studied enzyme was 155 U/mg protein with a purification degree of 67 and a yield of 2%. The peroxisomal form of MDH was characterized by a specific activity of 180 U/mg protein, the purification degree of 78, and a yield of 1%. An insignificant effect of 30 min incubation of the enzyme at 7, 15, and 30оС was demonstrated. Temperature optima for the direct and reverse reactions of three malate dehydrogenase isoforms were determined: 25 and 45оС for cytoplasmic, 30 and 45оС for mitochondrial isoform. The temperature optimum of the peroxisomal form for the reactions of malate oxidation and oxaloacetate reduction was the same, 35оС.
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