Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes.
We have isolated a full-length cDNA for human carbonic anhydrase IV (CA IV) from a lambda gt10 human kidney cDNA library. The 1105-base-pair (bp) cDNA contains a 47-bp 5' untranslated region, a 936-bp open reading frame, and a 122-bp 3' untranslated region. The deduced amino acid sequence is colinear with the N-terminal sequence and the sequence of several tryptic peptides of human lung CA IV. It includes an 18-amino acid signal sequence, a 260-amino acid region that shows 30-36% similarity with the 29-kDa cytoplasmic CAs (CA I, CA II, and CA III), and an additional 27-amino acid C-terminal sequence that ends in a 21-amino acid hydrophobic domain. Of the 17 "active site" residues that are highly conserved in other human CAs, 16 are also present in CA IV. Expression of the cDNA in COS cells produced a 35-kDa enzyme that was membrane associated, resistant to inactivation by SDS, contained no carbohydrate, and reacted on Western blots with antiserum to the 35-kDa CA IV from human lung. Treatment of membranes from transfected COS cells with phosphatidylinositol-specific phospholipase C released 20-30% of the expressed enzyme from membranes, indicating that at least 20-30% of the expressed enzyme was anchored to membranes by a glycosyl-phosphatidylinositol linkage.
Okuyama
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104.
Sato
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Zhu
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Waheed
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Sly
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D016415:Sequence Alignment; D012995:Solubility; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D006026:Glycoside Hydrolases; D002256:Carbonic Anhydrases; D043265:Phosphatidylinositol Diacylglycerol-Lyase