Sublethal ammonia and urea concentrations inhibit rainbow trout ( Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

Abstract

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout ( Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP +/min)/mg protein and ∼1600-fold in a yield of ∼60% by ammonium sulphate precipitation and 2′,5′-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2–5.5 μM) and urea (20–50 μM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver–Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex ( K i) and 50% inhibitory values were 2.26 ± 1.21 and 2.86 ± 3.51 μM for ammonia and 18.69 ± 6.75 and 23.77 ± 4.58 μM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant ( p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.