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First published online May 5, 2005
Journal of Experimental Biology 208, 1951-1961 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01551
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Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss: comparative physiology and molecular evolution

A. J. Esbaugh1,*, S. F. Perry2, M. Bayaa2, T. Georgalis2, J. Nickerson2, B. L. Tufts1 and K. M. Gilmour3,{dagger}

1 Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6
2 Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5
3 Department of Biology, Carleton University, Ottawa, ON, Canada K1S 5B6



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  		Fig. 1. Nucleotide and deduced amino acid sequence of carbonic anhydrase (CA) from the rainbow trout gill. Sequence shown is coding region only, from start codon (underlined) to stop codon (asterisk) as determined through RACE (rapid amplification of cDNA ends).

 


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  		Fig. 2. Phylogenetic analysis of rainbow trout cytoplasmic carbonic anhydrase (TCAc) and other {alpha}-CA isozymes. The phylogenetic tree was constructed using neighbour joining analysis with support for nodes assessed using bootstrap analysis. The tree was ordered using mouse, rat and human CA VII as a monophyletic outgroup. Branches are drawn to scale with the length of 0.1 approximating replacement of 10% of the amino acids in the protein alignment (no Poisson correction for multiple hits).

 


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  		Fig. 3. Comparison of the active sites of trout cytoplasmic catalytic anhydrase (CA) with those of trout red blood cell CA (TCAb), dace CA, gar CA, concensus trout CA I, CA II and human CA VII. Identical amino acids are indicated by a dot. aa diff, number of amino acid differences. *, putative active site; z, zinc binding ligand; +, proton shuttling associated ligand; ~, substrate associated pocket.

 


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  		Fig. 4. Representative northern blots for rainbow trout red blood cell carbonic anhydrase (TCAb), cytoplasmic carbonic anhydrase (TCAc) and ß-globin (Hb) mRNA and 18S rRNA from adult rainbow trout tissues (N=4). RBC, red blood cells; A, anterior; P, posterior.

 


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  		Fig. 5. Relative mRNA expression (mean ± S.E.M.) of cytoplasmic carbonic anhydrase (TCAc; A) and red blood cell carbonic anhydrase (TCAb; A) and haemoglobin (C) in rainbow trout tissues, as determined by real time RT-PCR (N=6). The red blood cell (RBC) mRNA expression was set to 1 in all cases.

 


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  		Fig. 6. The relative effect of plasma [which contains an endogenous carbonic anhydrase (CA) inhibitor] on the red blood cell (circles) and gill (squares) CA activity (mean ± S.E.M.) of rainbow trout (N=4). Significant differences are indicated by an asterisk (unpaired t-test; P<0.05).

 


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  		Fig. 7. The effects of anaemia on the relative mRNA expression (mean ± S.E.M.) of cytoplasmic carbonic anhydrase (TCAc) and red blood cell carbonic anhydrase (TCAb) in the red blood cells (A), and cytoplasmic carbonic anhydrase (TCAc) in gills (B) of rainbow trout, as determined by real time RT-PCR (N=6). Significant differences are indicated by an asterisk (P<0.05). Control values were set to 1 in all cases.

 

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© The Company of Biologists Ltd 2005