The use of phosphoenolpyruvate plus pyruvate kinase as an ATP-generating system in the assay for glutamine synthetase activity via the formation of gamma-glutamylhydroxamate from glutamate and hydroxylamine with crude tissue preparations is shown to give values far in excess of the true glutamine synthetase activity of the tissue. This is due to the generation of pyruvate, which reacts with hydroxylamine to give a compound that is chromogenic with the ferric chloride reagent used for measuring gamma-glutamylhydroxamate.
Rates of ammonia formation from six amino acids by hepatocytes and liver mitochondria were compared with the rate of ammonia excretion by individual fish. Glutamine and asparagine are the most ammoniagenic substrates for hepatocytes and glutamine and glutamate, for isolated liver mitochondria. The main site of ammonia release is the mitochondrion. Ammonia is formed mainly via deamidation or transdeamination. Relatively small amounts of aspartate are formed from glutamate and glutamine by the fish liver mitochondria. This apparent "uncoupling" of glutamate transamination may represent an adaptation of carnivorous fish to the utilization of amino acids as a major energy source. More than 90% of the ammonia formed from glutamate and glutamine by isolated mitochondria are elaborated into the medium. Exiting ammonia is accompanied by a proton, but the source of this proton appears not to be the electrogenic proton pump or the carboxylate of exiting glutamic acid.
The hepatic mechanism for detoxication of ammonia formed during amino acid gluconeogenesis in uricotelic vertebrates requires the intramitochondrial synthesis of glutamine by glutamine synthetase. This glutamine then serves as a precursor of uric acid in the cytosol. The evolutionary development of uricoteley thus required the localization of glutamine synthetase in liver mitochondria. The mechanism for the mitochondrial import of glutamine synthetase in uricotelic vertebrate liver is not yet known. Tortoises, extant relatives of the stem reptiles, possess both the ureotelic and uricotelic hepatic systems. It therefore seems likely that the genetic events allowing the mitochondrial localization of glutamine synthetase in liver occurred in the amniote amphibian ancestors of the stem reptiles. The selection of ureoteley by the theropsids and of uricoteley by the sauropsids were major events in the divergence and subsequent evolution of these two lines. Once established in the sauropsid line, uricoteley has persisted through to the higher reptiles, crocodilians, and birds. Uricoteley was in part responsible for the radiation of the archosaurs during the Triassic as a water-conserving mechanism in the adult, thereby allowing them to invade the arid environments of that period. Contrary to dogma, uricoteley was probably of minor significance in the development of the cleidoic egg. Neither mammalian nor avian embryonic liver tissues catabolize amino acids to any great extent, so it is inappropriate to attribute to them a kind of "waste" nitrogen metabolism.
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