Ways to Increase the Activity of Glutamate Dehydrogenase in Erythrocyte-Bioreactors for the Ammonium Removal

Abstract

An increased blood ammonium concentration (hyperammonemia) is toxic to the central nervous system, so removing excess ammonium from the bloodstream is an important task. One of the solutions may be the use of erythrocyte-bioreactors (EBRs) with ammonium-processing enzymes loaded inside. Earlier attempts have been made to create such EBRs based on glutamate dehydrogenase (GDH) or glutamine synthetase; however, these EBRs were not effective enough. We have previously shown that the reasons for this were the low permeability of the erythrocyte membrane for the substrates of these reactions (α-ketoglutarate and glutamate) and the low activity of the included GDH (due to its high molecular mass and tendency towards aggregation at an increase in concentration above 0.1 mg/mL), and also that the low membrane permeability problem for α-ketoglutarate and glutamate could be overcome if GDH and alanine aminotransferase were included into EBR together, because these metabolites should be consumed and reproduced within such EBR cyclically. However, the problem of low GDH activity remains if GDH is the main ammonium processing enzyme. To improve the efficiency of GDH incorporation into erythrocytes and the quality of the EBRs (their erythrocyte indices and osmotic fragility), these parameters were compared for various hypoosmotic methods for loading GDH into cells. In addition, a comparison was made of previously used GDH from bovine liver and GDH from Proteus sp. It was shown that the method of flow dialysis was the most effective and allowed the inclusion of the GDH amount 3 times exceeding the inclusion achieved during the hypoosmotic dialysis. The properties of erythrocytes after exposure to this method changed slightly. GDH from Proteus sp. did not aggregate with an increase in its concentration and thereby allowed an approximately 18-fold increase in the specific activity of the enzyme in erythrocytes. Thus, this GDH is a promising enzyme for creating effective EBRs for ammonium removing.