Biochemical characterization of a S-glutathionylated carbonic anhydrase isolated from gills of the Antarctic icefish Chionodraco hamatus.
Gill cytoplasmic carbonic anhydrase of the haemoglobinless Antarctic icefish Chionodraco hamatus (Ice-CA) was directly sequenced and consists in 259 residues with an acetylated N-terminus. The molecular mass, deduced from the sequence, was 28.45 kDa, while mass spectrometry analysis of the native protein gave higher values. Treatment with dithiothreitol abolished this difference, indicating possible post-translational modifications. Isoelectric focusing analysis of Ice-CA suggested S-thiolation, which was identified as S-glutathionylation by immunostaining. Deglutathionylated Ice-CA maintained the anhydrase activity but showed higher susceptibility to hydrogen peroxide, suggesting that glutathione binding to Cys residues may have a role in the defence against oxidative damage. Ice-CA is characterized by lower thermal stability, higher activity and lower activation energy than its homologue gill CA of the temperate European eel, confirming the adaptation of the catalytic capacity to low temperatures. Alignment of Ice-CA with homologous enzymes from other fish shows high identity; the enzyme is grouped with a previously described fish CA monophyletic clade although Ice-CA shows several characteristics that can increase protein-solvent interaction and structural flexibility.
Rizzello
Antonia
A
Laboratory of General Physiology, Department of Biological and Environmental Science and Technology, University of Salento, via Monteroni, 73100, Lecce, Italy.
Ciardiello
M Antonietta
MA
Acierno
Raffaele
R
Carratore
Vito
V
Verri
Tiziano
T
di Prisco
Guido
G
Storelli
Carlo
C
Maffia
Michele
M
D000595:Amino Acid Sequence; D000818:Animals; D000864:Antarctic Regions; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003080:Cold Temperature; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005399:Fishes / Q000235:genetics; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000502:physiology; D020539:Sequence Analysis, Protein
D029941:Fish Proteins; D016877:Oxidants; D006861:Hydrogen Peroxide; D002256:Carbonic Anhydrases; D005978:Glutathione