The physicochemical and catalytic properties of aconitate hydratase isozymes obtained from rat liver by chromatographic methods
Abstract
The aim of the study was to obtain homogeneous aconitate hydratase isozymes (aconitase, aconitate hydratase, EC 4.2.1.3) located in the mitochondria and cytoplasm of rat hepatocytes using chromatographic methods, and to study their physicochemical and catalytic properties. Aconitase activity was determined spectrophotometrically (at 240 nm). To obtain homogeneous preparations of aconitase, the enzyme was purified using chromatographic methods involving several steps (salting out the homogenate with ammonium sulphate, desalting or gel filtration using Sephadex G-25, ion exchange chromatography on DEAE Тоуореаrl, and gel chromatography on Sephadex G-200). The purity of the obtained enzyme preparations was assessed using polyacrylamide gel electrophoresis. The protein in the samples was measured according to Lowry. The molecular weight value of aconitase was determined by denaturing electrophoresis according to the Laemmle’s method. The universal protein staining was carried out using silver nitrate. In order to confirm that the isolated proteins corresponded to aconitate hydratase isozymes, we also carried out the specific gel development by tetrazolium method, adding NADP-isocitrate dehydrogenase (NADP-IDH) as a supporting enzyme.
Using chromatographic methods for purification of aconitase from the liver of Rattus norvegicus Wistar rats, we managed to obtain homogeneous aconitate hydratase enzymatic preparations with specific activity of cytosolic form of 1.736±0.024 U/mg of protein, and of mitochondrial form of 1.256±0.018 U/mg of protein. They were used for the study of physicochemical and kinetic characteristics. The molecular weight of cytoplasmic aconitase from rat hepatocytes was 91±5.2 kDa, and that of mitochondrial aconitase was 81±4.1 kDa. Both forms of aconitase were found to be homodimers. The molecular weight (Mr) of the subunits is 45 kDa for the cytoplasmic fraction and 41 kDa for the mitochondrial aconitase. The kinetic characteristics of the enzymatic reaction catalysed by the cytoplasmic and mitochondrial isozymes of aconitate hydratase obey the Michaelis-Menten equation. We determined that aconitase had a higher affinity to cis-aconitate than to citrate. It was shown that the isozyme from cytoplasm had a pH optimum of 8.0±0.1, i.e., was alkaline compared to the mitochondrial aconitase. (7.4±0.1).
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