Abstract
The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km app for acetyl coenzyme A increased while the Km app for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-acetylglutamate at the maximal rate.
Highlights
Ammonia is an obligatory waste product of protein catabolism that is highly toxic to the brain [1]
N-acetylglutamate synthase (NAGS), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), ornithine/citrulline transporter (ORNT) and citrin are expressed in the 32-cell embryos, suggesting that these mRNA are maternal
The relative expression of NAGS is higher in adult zebrafish than in embryos suggesting that NAGS could have a role in zebrafish physiology beyond ureagenesis
Summary
Ammonia is an obligatory waste product of protein catabolism that is highly toxic to the brain [1]. Adult fish excrete ammonia directly into water, urea cycle enzymes have been found in 23 species of fish [3]. Many fish are capable of ureagenesis and there are several fish species for which a need for the urea cycle can be explained. Since most fish rarely encounter water with high ammonia concentration [21], the need for ureagenesis in zebrafish and other fish is not clear. In these fish the urea cycle may be important for embryonic development. Zebrafish (Danio rerio), Atlantic cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss) embryos excrete most of their nitrogen waste as urea [22,23,24,25]. Expression of all enzymes and transporters required for the function of urea cycle were not measured in these studies
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