Pathways for urea production during early life of an air-breathing teleost, the African catfish Clarias gariepinus Burchell
Bendik F. Terjesen1,*,
Terry D. Chadwick2,
Johan A. J. Verreth3,
Ivar Rønnestad1 and
Patricia A. Wright2
1 Department of Zoology, University of Bergen, Allègaten, 41, N-5007 Bergen, Norway,
2 Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and
3 Fish Culture and Fisheries Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands
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Fig. 1. Individual wet mass (A) and relative water content (B) of whole embryos and yolk-sac larvae (filled triangles), dissected larval bodies (yolk-free animal) (open triangles), starved larvae (open circles), fed larvae (filled circles) and selected adult tissues (columns) of Clarias gariepinus. Note that wet mass of fed larvae refers to the right-hand axis in A. Values are means ± S.D. of 8–9 samples (A) or three samples (B), except at 0h post-fertilization (N=2, ± range). CYA, complete yolk absorption.
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Fig. 2. Individual urea-N content (A) and concentration (B) of whole embryos and yolk-sac larvae (filled triangles), dissected larval bodies (yolk-free animal) (open triangles), starved larvae (open circles), fed larvae (filled circles) and selected adult tissues (columns) of Clarias gariepinus. Note that urea-N content of fed larvae refers to the right-hand axis in A. Values are means ± S.D. of three samples. CYA, complete yolk absorption.
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Fig. 3. Uricase (A), allantoinase (B), allantoicase (C) and ureidoglycollate lyase (UGL, D) activity in whole-animal crude extracts during early development and in selected adult tissues (columns) of Clarias gariepinus. Filled triangles, whole embryos and yolk-sac larvae; filled circles, larvae fed Artemia; open circles, starved larvae. Values are means ± S.D. of assays on 3–6 samples for uricase and allantoinase. Allantoicase and UGL activity are the means ± S.D. of three samples, except at 54, 99, 120, 170 194, 217 and 313h post-fertilization for fed larvae (N=2, ± range). CYA, complete yolk absorption; ND, not detectable.
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Fig. 4. Elution profile of fed Clarias gariepinus larval extracts subjected to gel filtration chromatography on a Sephacryl S300HR column and assayed for carbamoyl phosphate synthetase (CPSase) activity as described in the text. Note differences in scaling of the axes for the larval liver elution. Whole larvae (313h post-fertilization) elution: flow rate 0.31mlmin-1, sample volume 3.5ml, protein concentration 7.3mgml-1. Larval muscle tissue (217h post-fertilization): flow rate 0.30mlmin-1, sample volume 2.4ml, protein concentration 3.9mgml-1. Larval liver tissue (217h post-fertilization): flow rate 0.33mlmin-1, sample volume 1.9ml, protein concentration 1.6mgml-1. hPF, hours post-fertilization; CP, carbamoyl phosphate.
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Fig. 5. Effects of varying glutamine (Gln) concentration on carbamoyl phosphate synthetase III (CPSase III) activity in Clarias gariepinus larval extracts (313h post-fertilization) with or without N-acetyl-L-glutamate (AGA) present in the reaction mixture. The enzyme was obtained from gel chromatography (Fig.4, whole larvae) by pooling fractions from the second peak having more than 60% of maximum peak activity (protein concentration 0.59mgml-1). Assays were conducted using standard conditions as described in the text, except as noted. The inset is a reciprocal plot of activity (V) versus substrate concentration. Because of the high dependence on AGA of this CPSase III, activity was too low and variable to estimate Km in the absence of the effector. CP, carbamoyl phosphate.
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Fig. 6. Carbamoyl phosphate synthetase II (CPSase II) (A) and CPSase III (B) activity in whole-animal crude extracts during early development and in selected adult tissues (columns) of Clarias gariepinus. Filled triangles, whole embryos and yolk-sac larvae; filled circles, larvae fed Artemia; open circles, starved larvae.Values are means ± S.D. of assays on 3–4 samples, except for fed larvae at 79 and 119h post-fertilization (N=2, ± range). CPSase II activity was calculated from the rate obtained with glutamine (Gln) only in the reaction mix, while CPSase III was calculated from the rate with Gln + N-acetyl-L-glutamate (AGA) minus the rate with Gln only. The relative capacities of the two CPSases in total carbamoyl phosphate formation in vitro are shown in C (% UTP inhibition calculated as described in Table1, high values: mostly CPSase II) and D (AGA activation factor, rate with Gln + AGA/rate with Gln only, high values: mostly CPSase III). CYA, complete yolk absorption; ND, not detectable.
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Fig. 7. Glutamine synthetase (GSase, A), ornithine carbamoyl transferase (OCTase, B) and arginase (C) activity in whole-animal crude extracts during early development and in selected adult tissues (columns) of Clarias gariepinus. Filled triangles, whole embryos and yolk-sac larvae; filled circles, larvae fed Artemia; open circles, starved larvae. Values are means ± S.D. of assays on 3–4 samples, except for fed larvae at 79 and 119h post-fertilization (N=2, ± range). CYA, complete yolk absorption.
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© The Company of Biologists Ltd 2001
