Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase.
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 approximately 1600-fold in a yield of approximately 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 microM) and urea (20-50 microM) 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 (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM 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.
Erdoğan
Orhan
O
Department of Aquaculture, Agriculture Faculty, Atatürk University, 24240 Erzurum, Turkey.
Hisar
Olcay
O
Köroğlu
Günay
G
Ciltaş
Abdulkadir
A
D000641:Ammonia / Q000633:toxicity*; D000818:Animals; D004912:Erythrocytes / Q000187:drug effects* / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors*; D017686:Oncorhynchus mykiss; D018384:Oxidative Stress; D014508:Urea / Q000633:toxicity*
D000641:Ammonia; D014508:Urea; D005954:Glucosephosphate Dehydrogenase