Regulation of N-acetylglutamate levels through glutaminase activity : a potential mediator of urea synthesis

Ball, Stephen William Damian (2003) Regulation of N-acetylglutamate levels through glutaminase activity : a potential mediator of urea synthesis. Masters thesis, Memorial University of Newfoundland.

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Abstract

N-Acetylglutamate synthetase catalyses the reaction of glutamate and acetyl-CoA to produce N-acetylglutamate (NAG), which is an essential activator of carbamoyl phosphate synthase I (CPS I). CPS I is the flux-controlling enzyme of the urea cycle. Hepatic glutaminase is believed to be associated with the urea cycle but the nature of this association is not fully understood. Our hypothesis is that hepatic glutaminase provides glutamate for N-acetylglutamate synthesis. Therefore, factors which increase glutaminase activity should activate CPS I by increasing NAG levels. -- We investigated the effects of the known activators of glutaminase, glucagon and NH₄C1, in perfused rat livers. Mitochondria were subsequently prepared from these livers and NAG levels and citrulline synthesis assessed. Both glucagon (1O⁻⁷M) and NH₄C1 (1 mM) increased mitochondrial NAG levels and the rate of citulline synthesis. This was in agreement with previous results. -- We proceeded to investigate whether a link between glutaminase activity and NAG levels existed. We employed 6-diazo-5-oxo-norleucine, DON, a compound we found to be a suitable inhibitor of glutaminase activity. Glucagon significantly increased NAG and citrulline synthesis in mitochondria prepared from hepatocyte incubations. However, pre-incubation of hepatocytes with DON significantly decreased the stimulatory effect of glucagon on NAG levels and subsequent citrulline synthesis. Hepatocyte incubations with 1 mM ammonia also yielded a significant increase in mitochondrial NAG levels. Once again, pre-incubation of hepatocytes with DON significantly inhibited the effect mediated by ammonia on mitochondrial NAG content. These results suggest a link between glutaminase activation and mitochondrial levels of NAG. -- The increase in mitochondrial NAG due to ammonia stimulation, however, did not correlate with an increase in the rate of citrulline synthesis. Therefore, there may exist some means by which mitochondria down regulate the activation of citrulline synthesis despite elevated levels of NAG. The mechanism by which the kinetic relationship between NAG levels and citrulline synthesis is disrupted remains unknown. Our results have led us to suggest that glutaminase may indirectly affect urea production through its effects on NAG levels.

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