Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney.
We have purified carbonic anhydrase (CA) IV from human lung membranes to apparent homogeneity in a form which is catalytically active and stable to storage. It has an apparent molecular mass of 35 kDa, is insensitive to endoglycosidases, and seems to contain no N-linked or O-linked oligosaccharide chains. Reduction of disulfide linkages led to altered migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and loss of catalytic activity. CA IV resembles CA II in being a "high activity" isozyme, relatively resistant to inhibition by halide ions and sensitive to inhibition by sulfonamides. Application of this purification to human kidney membranes produced homogeneous enzyme with nearly identical properties. Amino acid compositions of both lung and kidney CA IV were similar, as were tryptic peptide patterns resolved on high performance liquid chromatography (HPLC). Amino-terminal sequences of native enzyme from lung and kidney were identical, as were amino-terminal sequences of the three major tryptic peptides resolved on reverse phase HPLC. Isoelectric focusing revealed microheterogeneity in enzyme from both sources. Antibody raised to human lung CA IV reacted equally strongly with CA IV from kidney, but very weakly or not at all with other CAs. Treatment of lung membranes and kidney membranes with phosphatidylinositol-specific phospholipase C released over half of the membrane-bound CA IV, suggesting that at least half of the CA IV in both organs is anchored to membranes by phosphatidylinositol-glycan linkages.
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
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D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin
D007527:Isoenzymes; D009844:Oligosaccharides; D010446:Peptide Fragments; D014357:Trypsin; D002256:Carbonic Anhydrases