The use of malate dehydrogenase preparation, obtained by the chromatographic method, for the specific staining of fumarate hydratase from rat hepatocytes
Abstract
The aim of this study was to obtain a highly purified preparation of malate dehydrogenase (MDH) by chromatographic methods for subsequent specific staining of fumarate hydratase (fumarase, FH) from the liver of healthy rats and animals with alloxan-induced diabetes. The first stage of the study was the performance of a multi-stage purification scheme for MDH using ion-exchange chromatography as the final stage, which resulted in the obtaining of two enzyme preparations, from which a sample with the highest activity (with a yield of 20% and a specific activity of 32.5 U/mg protein) was selected. MDH activity was measured spectrophotometrically at a wavelength of 340 nm, protein concentration was determined by the Lowry method. Experimental type 1 diabetes mellitus was induced by a single intraperitoneal injection of 5% alloxan monohydrate solution in 0.9% sodium citrate solution to male white inbred laboratory Wistar rats (Rattus norvegicus L). The animals were kept in a vivarium with constant access to food and water. The conditions of the experiment corresponded to the requirements of international rules for the humane treatment of animals, reflected in the sanitary rules for the selection and maintenance of experimental biological clinics (vivariums). All rats were randomly divided into 2 groups (Control and Diabetes), 5 rats in each group.
Fumarate hydratase is a lyase and it is not an oxidizing enzyme; therefore, it cannot be specifically detected in polyacrylamide gel by the tetrazolium method. However, this specific staining technique can also be used for FH by adding malate dehydrogenase as an auxiliary enzyme to the development medium. Polyacrylamide gel electrophoresis was performed under non-denaturing conditions in Tris-glycine buffer. Conducted electrophoretic studies with subsequent staining of the gel with the tetrazolium method showed that in the liver of both healthy rats and animals with pathology, two isoforms of fumarate hydratase with Rf 0.12 and 0.2 respectively, were present. An analysis of the subcellular localization of FH showed that the identified forms of the enzyme function in the cytoplasm and mitochondria of the liver of rats of both groups. Thus, the use of ion-exchange chromatography as the main step allowed to obtain a highly purified preparation of malate dehydrogenase, which was later used as an auxiliary enzyme in the specific staining of fumarate hydratase from the liver of rats with alloxan-induced diabetes. The obtained results indicate that the development of type 1 diabetes mellitus, unlike some types of oncology, is not associated with blocking of one of FH isoforms.
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