| Summary: | Aim: A cytosolic carbonic anhydrase (I-CA) isoform, showing higher catalytic rate with respect to temperate counterparts, was isolated from the gills of the haemoglobinless, circulating CA deprived Antarctic teleost Chionodraco hamatus. To understand the molecular basis of the high I-CA activity, a structural characterisation has been undertaken. Methods: I-CA was purified from gill epithelium by sulphanilamide sepharose gel FPLC. The amino acid sequence of I-CA was established by direct protein sequencing. Isoelectric focusing (IEF) and Western blot analysis were used to show post-translational modifications of I-CA. Thermodynamic parameters of CA were calculated by measuring carbonic anhydrase activity in the 0-40 °C range by a radioactive assay. Sequence analysis and computational procedures were performed. Results: I-CA consists of 259 residues and its molecular mass deduced from the amino acid sequence (28449 Da) was higher than the value obtained by mass spectrometry (2873829225 Da). Treatment with dithiothreitol (DTT) abolished this discrepancy, indicating possible post-translational modifications also confirmed by IEF analysis. S-glutathionylation of I-CA was demonstrated by using specific anti-GSH antibody. Deglutathionylated form of I-CA maintained its activity despite a higher susceptibility to H2O2. Thermodynamic parameters of I-CA showed some traits of cold-adaptation as described for other enzymes from Antarctic fish. The I-CA sequence also suggests specific adaptive features related to the environmental temperature. Conclusion: A novel enzyme variant with high turnover rate was found in the respiratory epithelium of the icefish. The evolution of this branchial isoform could correlate with cold adaptation and the absence of haemoglobin and circulating CA in this fish.
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