Subcellular localization and biochemical properties of the enzymes of carbamoyl phosphate and urea synthesis in the batrachoidid fishes Opsanus beta, Opsanus tau and Porichthys notatus

1995 ◽  
Vol 198 (3) ◽  
pp. 755-766 ◽  
Author(s):  
P Walsh
Keyword(s):  
Glutamine Synthetase ◽  
Subcellular Localization ◽  
Biochemical Properties ◽  
Nitrogen Excretion ◽  
Urea Synthesis ◽  
Carbamoyl Phosphate ◽  
Opsanus Beta ◽  
Opsanus Tau ◽  
Porichthys Notatus ◽  
The Family

The subcellular localization and biochemical properties of the enzymes of carbamoyl phosphate and urea synthesis were examined in three representatives of fishes of the family Batrachoididae, the gulf toadfish (Opsanus beta), the oyster toadfish (Opsanus tau) and the plainfin midshipman (Porichthys notatus). The primary objective of the study was to compare the biochemical characteristics of these fishes, which represent a range between ammoniotelism and ureotelism (O. beta being facultatively ureotelic), with previous patterns observed for an ammoniotelic teleost (Micropterus salmoides, the largemouth bass) and an obligate ureogenic elasmobranch (Squalus acanthias, the dogfish shark). The present study documents the expression of mitochondrial carbamoyl phosphate synthetase (CPSase) III and cytosolic CPSase II (and its associated enzymes of pyrimidine synthesis, dihydro-orotase and aspartate carbamoyltransferase) in the livers of all three batrachoidid species. Both mitochondrial and cytosolic activities of arginase were present in the livers of all three species, as were cytosolic glutamine synthetase and argininosuccinate synthetase and lyase. However, O. beta also showed mitochondrial glutamine synthetase activity and higher total hepatic levels of glutamine synthetase than either O. tau or P. notatus. Taken together, these observations confirm that the arrangement of these enzymes in the batrachoidid fishes has greater similarity to that of M. salmoides than to that of S. acanthias. However, differences within the family appear to coincide with the different nitrogen excretion strategies. O. tau and P. notatus are primarily ammoniotelic and most closely resemble the ammoniotelic M. salmoides, whereas ureotelism in O. beta is correlated with the presence of a mitochondrial glutamine synthetase and the ability to induce higher total glutamine synthetase activities than O. tau or P. notatus. Additionally, isolated mitochondria from O. beta were able to generate citrulline from glutamine, whereas those from O. tau were not. Also in contrast to S. acanthias, glutamine synthetase activities in the mitochondria of O. beta are consistently lower than those of CPSase III. This and other kinetic observations lend support to the hypothesis that glutamine synthetase may be an important regulatory control point in determining rates of ureogenesis in O. beta.

scholarly journals Induction of carbamoyl phosphate synthetase III and glutamine synthetase mRNA during confinement stress in gulf toadfish (Opsanus beta)

2000 ◽  
Vol 203 (2) ◽  
pp. 311-320 ◽  
Author(s):  
H. Kong ◽  
N. Kahatapitiya ◽  
K. Kingsley ◽  
W.L. Salo ◽  
P.M. Anderson ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Waste Product ◽  
Mrna Levels ◽  
Urea Synthesis ◽  
Carbamoyl Phosphate ◽  
Confinement Stress ◽  
Actin Mrna ◽  
Mrna Analysis ◽  
Ribonuclease Protection ◽  
Gulf Toadfish

Gulf toadfish (Opsanus β) rapidly switch to excretion of urea as their main nitrogenous waste product under several laboratory conditions, including confinement to small volumes of water. Prior evidence suggested that the activities of two key enzymes of urea synthesis exhibited potentially different modes of upregulation during this switch, with carbamoyl phosphate synthethase III (CPSase III) activated allosterically by N-acetylglutamate, and glutamine synthetase (GSase) activated by increases in the concentration of protein. The present study was undertaken to examine additional aspects of the regulation of these enzymes. The sequence for O. beta CPSase III cDNA was obtained, and it was found to be similar to that of other piscine CPSases. The sequence also allowed us to develop riboprobes for CPSase III mRNA analysis using ribonuclease protection assays (RPAs). CPSase III mRNA was expressed in liver, muscle, kidney and intestine, in agreement with prior enzymatic measurements. Levels of CPSase III mRNA increased five- to tenfold (relative to beta-actin mRNA) in liver (but not muscle) following 48 h of confinement stress. Measured by western analysis using an antibody to chicken GSase, confined O. beta GSase protein concentrations increased eightfold over control levels, in agreement with prior and present measurements of increases in GSase activity. Furthermore, RPAs of GSase mRNA levels demonstrated an increase of fivefold during confinement.

scholarly journals EFFECTS OF CONFINEMENT/CROWDING ON UREOGENESIS IN THE GULF TOADFISH OPSANUS BETA

1994 ◽  
Vol 191 (1) ◽  
pp. 195-206 ◽  
Author(s):  
P Walsh ◽  
B Tucker ◽  
T Hopkins
Keyword(s):  
Amino Acid ◽  
Glutamine Synthetase ◽  
Nitrogen Excretion ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Opsanus Beta ◽  
Aspartate Amino Transferase ◽  
Biochemical Mechanisms ◽  
Amino Acid Concentrations ◽  
Gulf Toadfish

In order to elucidate the cues for, and the biochemical mechanisms of, the transition to ureogenesis in the gulf toadfish Opsanus beta, experiments on the effects of confinement/crowding were carried out. Confinement of toadfish to small volumes of water initiated a switch to nearly complete reliance on ureogenesis for nitrogen excretion within 24­48 h. Further experiments suggested that this switch was probably due to the physical confinement per se, rather than to a measurable build-up of ammonia in the water. However, the possibility of the response being triggered by a very low concentration of a pheromone-like substance was not excluded by our experimental design. The activities of several enzymes of ureogenesis, ornithine­citrulline transcarbamoylase, aspartate amino transferase and glutamine synthetase, increased in the liver of toadfish during confinement. Notably, glutamine synthetase activity increased almost fourfold within 24 h, and this increase preceded increases in urea excretion. A number of plasma and liver amino acid concentrations changed during confinement: there were declines in plasma asparagine, glutamate and glycine levels and an increase in plasma valine, as well as a decline in liver alanine and an increase in liver arginine concentrations. Liver glutamine was not detectable. When the amino acid data are taken together with the enzyme activity changes, it appears that the switch to ureogenesis occurs primarily upstream of the ornithine­urea cycle, at the level of supply of nitrogen to the pathway. The results are discussed in the context of the habitat of toadfish.

scholarly journals Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish)

1989 ◽  
Vol 261 (2) ◽  
pp. 523-529 ◽  
Author(s):  
P M Anderson
Keyword(s):  
Glutamine Synthetase ◽  
Enzyme Activities ◽  
Squalus Acanthias ◽  
Spiny Dogfish ◽  
Urea Synthesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Pyrimidine Nucleotide ◽  
Aspartate Carbamoyltransferase ◽  
Pyrimidine Pathway

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5′-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.

scholarly journals Ureotelism is inducible in the neotropical freshwater Hoplias malabaricus (Teleostei, Erythrinidae)

2004 ◽  
Vol 64 (2) ◽  
pp. 265-271 ◽  
Author(s):  
G. Moraes ◽  
V. L. P. Polez
Keyword(s):  
Specific Activity ◽  
Excretion Rate ◽  
Nitrogen Excretion ◽  
Urea Synthesis ◽  
Plasma Ammonia ◽  
Alkaline Water ◽  
Hoplias Malabaricus ◽  
Urea Excretion ◽  
Ammonia Transport ◽  
In The Wild

Increased environmental pH decreases ammonia transport through the gills, impairing nitrogenous waste. The consequent toxicity is usually drastic to most fishes. A few species are able to synthesize urea as a way to detoxify plasma ammonia. We studied three teleosts of the family Erythrinidae living in distinct environments, and assumed the biochemical behaviors would be different in spite of their being closely related species. Adult fish collected in the wild were submitted to alkaline water and the urea excretion rate was determined. The specific activity of urea cycle enzymes was determined in liver samples of fish from neutral waters. The studied species Hoplias lacerdae, Hoplerithrynus unitaeniatus, and Hoplias malabaricus are ureogenic. Urea synthesis is not a metabolic way to detoxify ammonia in H. lacerdae and Hoplerithrynus unitaeniatus exposed to an alkaline environment. The plasma ammonia profile of both species showed two distinct biochemical responses. Urea excretion of H. malabaricus was high in alkaline water, and the transition to ureotelism is proposed. The nitrogen excretion rate of H. malabaricus was among the highest values reported and the high urea excretion leads us to include this species as ureotelic in alkaline water.

scholarly journals Widespread use of emersion and cutaneous ammonia excretion in Aplocheiloid killifishes

2018 ◽  
Vol 285 (1884) ◽  
pp. 20181496 ◽  
Author(s):  
Michael D. Livingston ◽  
Vikram V. Bhargav ◽  
Andy J. Turko ◽  
Jonathan M. Wilson ◽  
Patricia A. Wright
Keyword(s):  
Ammonia Excretion ◽  
Nitrogen Excretion ◽  
Gaseous Ammonia ◽  
Urea Synthesis ◽  
Air Exposure ◽  
Ammonia Accumulation ◽  
Common Strategy ◽  
Kryptolebias Marmoratus ◽  
Amphibious Fishes ◽  
New Strategies

The invasion of land required amphibious fishes to evolve new strategies to avoid toxic ammonia accumulation in the absence of water flow over the gills. We investigated amphibious behaviour and nitrogen excretion strategies in six phylogenetically diverse Aplocheiloid killifishes ( Anablepsoides hartii, Cynodonichthys hildebrandi, Rivulus cylindraceus , Kryptolebias marmoratus, Fundulopanchax gardneri , and Aplocheilus lineatus ) in order to determine if a common strategy evolved . All species voluntarily emersed (left water) over several days, and also in response to environmental stressors (low O 2 , high temperature). All species were ammoniotelic in water and released gaseous ammonia (NH 3 volatilization) during air exposure as the primary route for nitrogen excretion. Metabolic depression, urea synthesis, and/or ammonia accumulation during air exposure were not common strategies used by these species. Immunostaining revealed the presence of ammonia-transporting Rhesus proteins (Rhcg1 and Rhcg2) in the skin of all six species, indicating a shared mechanism for ammonia volatilization. We also found Rhcg in the skin of several other fully aquatic fishes, implying that cutaneous ammonia excretion is not exclusive to amphibious fishes. Overall, our results demonstrate that similar nitrogen excretion strategies while out of water were used by all killifish species tested; possibly the result of shared ancestral amphibious traits, phenotypic convergence, or a combination of both.

scholarly journals Biochemical properties and subcellular localization of six members of the HXK family in maize and its metabolic contribution to embryo germination

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Giovanna Paulina Aguilera-Alvarado ◽  
Ángel Arturo Guevara-García ◽  
Samuel Abraham Estrada-Antolín ◽  
Sobeida Sánchez-Nieto
Keyword(s):  
Subcellular Localization ◽  
Biochemical Properties ◽  
Embryo Germination
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