Membrane-associated carbonic anhydrase from the crab gill: purification, characterization, and comparison with mammalian CAs.
Carbonic anhydrase (CA) was purified from the gills of the shore crab Carcinus maenas using affinity chromatography and HPLC. The predominantly membrane-bound CA was found to share several features with mammalian CA IV. Its apparent molecular weight of 36 kDa was reduced to 33 kDa by treatment with PNGase F, suggesting that crab CA is a glycoprotein with one N-linked oligosaccharide chain. More than half of the membrane-bound crab CA was released from membranes by treatment with a phosphatidylinositol-specific phospholipase C, indicating that the branchial CA is anchored to membrane surfaces by a phosphatidylinositol-glycan linkage. The enzyme also resembles mammalian CA IV in its relative sensitivity to inhibition by sulfonamides and the resistance to inhibition by halide ions. Amino acid composition of the HPLC-purified crab CA was examined and CNBr cleavage was carried out followed by N-terminal amino acid sequencing. Amino-terminal sequence of the native enzyme differed considerably from those of mammalian isozymes (human CA I and CA II, bovine CA III, human and rat CA IV). However, antisera raised against rat CA IV, CA II, and CA I all cross-reacted weakly with crab CA. Unlike mammalian CA IVs, crab gill CA was sensitive to 0.2% sodium dodecyl sulfate, suggesting that although crab gill CA is like mammalian CA IVs in many ways, it is less stabilized by intramolecular disulfide bonds.
Böttcher
K
K
Biologische Anstalt Helgoland, Hamburg, FRG.
Waheed
A
A
Sly
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D003386:Brachyura / Q000201:enzymology*; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification*; D003429:Cross Reactions; D003488:Cyanogen Bromide; D003600:Cytosol / Q000201:enzymology; D005880:Gills / Q000201:enzymology*; D006023:Glycoproteins / Q000302:isolation & purification; D007527:Isoenzymes; D008168:Lung / Q000201:enzymology; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments; D051381:Rats; D017386:Sequence Homology, Amino Acid
D002257:Carbonic Anhydrase Inhibitors; D006023:Glycoproteins; D007527:Isoenzymes; D010446:Peptide Fragments; D002256:Carbonic Anhydrases; D003488:Cyanogen Bromide