Carbamoyl phosphate synthetase (CPS) from E. coli catalyzes the formation of carbamoyl phosphate, an intermediate in the biosynthesis of pyrimidine nucleotides and arginine, from glutamine, bicarbonate and two molecules of MgATP. This reaction is catalyzed by three separate active sites that are separated in space by ~100 Å. The transfer of ammonia and carbamate through the two intramolecular tunnels was investigated by molecular dynamics simulations and experimental characterization of mutations within. The presence of an unstable reaction intermediate, carboxyphosphate, was established. A method for studying the synchronization of the two active sites on the large subunit of CPS was developed. The potential of mean force (PMF) calculations along the ammonia and carbamate transfer pathways indicate a low free-energy path for the translocation of ammonia. The highest barrier for ammonia is 7.2 kcal/mol which corresponds to a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. A blockage in the passageway was introduced by the triple mutant C232V/A251V/A314V, which was unable to synthesize carbamoyl phosphate. The release of phosphate is necessary for the injection of carbamate into the carbamate tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through carbamate tunnel. The mutants retained only 1.7 percent and 3.8 percent of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild-type CPS, respectively. Formate can be utilized by CPS in the absence of bicarbonate to form formyl phosphate. This intermediate was observed by 31P, 13C, and 1H NMR. For the three NMR methods a peak corresponding to formyl phosphate was observed at 2.15 ppm (31P) , 162.4 ppm (13C), and 8.39 and 7.94 ppm (1H). The rate of formation of formyl phosphate is 0.025 ± 0.005 s-1. Formamide was not detected in the presence of an ammonia source. Fluorescence anisotropy measurements on the C551A/S171C and C551A/S717C mutants provided insight into a possible mechanism of synchronization between the two active sites on the large subunit. The biggest fluorescence anisotropy change was observed at the N-terminal domain in the presence of AMPPNP and ATP.

Ammonia excretion from the freshwater fish gill is thought to occur mainly via passive diffusion of NH3 aided by a favourable plasma-to-water ammonia gradient sustained by a pH gradient formed by an acidified gill boundary layer. Rhesus (Rh) proteins are the newest members of the ammonia transporter superfamily. In this thesis research, ten rainbow trout Rh cDNA sequences were cloned and characterized. Rhcg2 mRNA and H+-ATPase mRNA and activity levels were upregulated in the trout gill pavement cells in response to experimentally elevated plasma ammonia, concurrent with enhanced ammonia excretion. Controversially, Rh proteins are thought to transport C02. However, Rh mRNA levels in most tissues of hypercapnia-exposed trout remained stable suggesting that trout Rh proteins likely do not conduct C02. Xenopus oocytes expressing trout Rh proteins facilitated the bi-directional transport of methylamine, an ammonia analogue. Methylamine transport was inhibited by ammonia and sensitive to a pH gradient and the concentration of the protonated species. Use of the scanning ion electrode technique (SIET) indicated that trout Rh proteins have an ammonia affinity within the physiological range, which is greater than that for methylamine, and they transport ammonia more rapidly than methylamine. A model of ammonia excretion in the trout gill pavement cell is proposed wherein ammonia enters via basolateral Rhbg and exits via apical Rhcg2, binding to these channels as NH4+ but transiting as NH3. In the gill boundary layer, NH3 combines with an H+ ion released from H+-ATPase and/or Na+/H+ exchange, forming NH4+. As low-affinity, high-capacity ammonia transporters, Rh proteins in the trout gill would exploit the favourable pH gradient formed by the acidic boundary layer to facilitate rapid ammonia efflux when plasma ammonia levels are elevated. Basal plasma ammonia levels are likely maintained by simple passive NH3 diffusion with a smaller role for Rh proteins under these conditions. / Thesis / Doctor of Philosophy (PhD)

<p>Ammonia is the primary nitrogenous waste product in teleost fish, which is produced primarily through protein metabolism. Fish experience natural elevations in internal ammonia loads, including during digestion where luminal ammonia concentrations in the intestine rise substantially. Furthermore, the intestine may absorb a portion of this ammonia, despite it being toxic to the fish. Based on this, <em>in vitro </em>techniques were employed in order to develop a model for teleost intestinal ammonia handling.</p> <p>Ammonia absorption and endogenous ammonia production occur along the entire length of the intestine. However, section-specific differences exist in terms of both endogenously produced ammonia and ammonia flux rates, with the highest rates in the anterior and mid intestine. Feeding stimulated an increase in production rates in all intestinal sections. Overall, ammonia originating from the gut may account for up to 42% of post-prandial whole-fish ammonia excretion. This could partly be attributed to the increased activity of the ammonia-producing enzyme glutamate dehydrogenase, and decreased activity of the ammonia-fixing glutamine synthetase. Furthermore, gut tissue ammonia concentrations surpassed typical chyme concentrations and were well regulated independent of high luminal ammonia, suggesting active transport across the intestinal epithelium.</p> <p>Seawater (60%) acclimation caused no substantial changes in the ammonia handling properties of the intestine. Ammonia transport in the intestine of both freshwater and seawater trout appears to occur via active means, coupled to Na⁺/K⁺ ATPase activity. Specifically, this involves Na⁺ linked transport through substitution of NH₄⁺ for K⁺on the apical Na⁺/K⁺/2Cl^- co-transporter occurring predominantly in the anterior and mid intestine, and solvent drag through fluid transport (osmotically driven by active NaCl absorption) in all sections. Additionally, Rhesus glycoprotein mediated ammonia transport likely occurs through basolateral Rhbg1, supporting previous molecular evidence. Overall this thesis illuminates the quantitative importance and mechanisms of gut ammonia transport in fish, and highlights future research avenues.</p> / Master of Science (MSc)

Teleost fishes incorporate renal ammonia excretion as part of a greater acid-base regulatory system. However, the transport mechanisms employed by the renal epithelium to excrete ammonia are relatively unknown. I hypothesized that, under metabolic acidosis, increased renal ammonia excretion would be the product of tubular secretion and involve a Na+/NH4+ exchange metabolon mediated through Rhesus (Rh) glycoproteins. To induce metabolic acidosis, goldfish (Carassius auratus) were exposed to a low pH environment (pH 4.0; 48-h). There was a clear signal of metabolic acidosis: a reduction in both plasma [HCO3-] and blood pH with no influence on plasma PCO2. Goldfish demonstrated an elevation in total plasma [ammonia] with a reduction in PNH3 under acidosis. Metabolic acidosis induced higher rates of urinary excretion of acidic equivalents in the form of both NH4+ and titratable acidity-HCO3- (TA-HCO3-) excretion. Urinary Na+ excretion was not affected by acidosis and urine [Na+] did not correlate with urinary [ammonia]. Alanine aminotransferase activity in the kidney was higher in acidotic goldfish. Glomerular filtration rate and urine flow rate were not affected by acidosis. Increased renal NH4+ excretion was due to increased secretion, and not increased filtration, of ammonia. There was a corresponding elevation in Rhcg1b mRNA expression but no change in renal Na+ reabsorption. My data support a secretion-based mechanism of teleost renal ammonia transport. This system is Na+ independent and is likely mediated by Rh glycoproteins and H+ ATPase, involving a parallel H+/NH3 secretion mechanism. To investigate effects of metabolic acidosis on elasmobranch fish, Pacific spiny dogfish (Squalus acanthias suckleyi) were infused with an acidic saline (125 mM HCl/375 mM NaCl; 3 ml/kg/h; 24-h). The results are preliminary, with no marked effects of HCl infusion on plasma acid-base or N-status, but increased branchial NHE2 and lower renal NHE3 protein expressions. These data are summarized in an Appendix. / Thesis / Master of Science (MSc)