Using ion exchange chromatography to obtain the lactate dehydrogenase of rat liver
Abstract
A purification scheme has been proposed, consisting of three successive stages, which makes it
possible to conduct a highly effective separation of forms of lactate dehydrogenase from rat liver. Obtainingisoenzymes in a highly purified state opens up prospects for the study of their catalytic and regulatory characteristics.
This is of particular importance for the future study of the functioning of this enzyme system in the
hepatocytes of rats with alloxan-induced diabetes. It is known that the adaptive reaction of cellular metabolism to experimental diabetes occurs at the level of enzyme systems of the Krebs cycle and the glyoxylate pathway.
The results of the study of regulatory and physico-chemical characteristics indicate that LDH isoenzymes
obtained from rat hepatocytes differ in electrophoretic mobility and affinity to the substrate and coenzyme.
The Rf values were 0.07 for LDH1, 0.16 for LDH2, and 0.25 for LDH3 . Earlier studies of this enzyme
in peas carried out by the researchers from our department have shown that the electrophoregram of this body with a specific staining shows a single band with Rf equal to 0.65.
The affinity of the enzyme towards the substrate is of great importance for the rate of enzyme catalysis.
Therefore, the determined catalytic characteristics allow us to justify the transformation of the way pyruvate is utilized in the cells with pathologies caused by experimental diabetes as compared to normal rat cells. The highest affinity to the substrate was observed in third isoenzymes (Km=0.227 mM) and the lowest in LDH1 (Km=14.104 mM). Interestingly, unlike the LDH of another mammal, the black-lipped pika, the two isoenzymes of rat LDH have a much higher affinity to pyruvate.
Thus, ion-exchange chromatography was used to obtain three lactate dehydrogenase isoforms from
rat liver cells and to study their physical and chemical characteristics.
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