title: Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum.
abstract: A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.
authors.affiliation: Department of Chemistry, Kwansei-Gakuin University, 1-1-155 Uegahara, Nishinomiya 662-8501, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Satoh
authors.forename: Y
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: B
authors.initials: B
authors.lastname: Colman
authors.forename: Y
authors.initials: Y
authors.lastname: Matsuda
mesh_terms: D020418:Algal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D017554:Carbon Compounds, Inorganic / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010788:Photosynthesis / Q000187:drug effects; D018749:RNA, Plant; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D020418:Algal Proteins; D017554:Carbon Compounds, Inorganic; D018076:DNA, Complementary; D018744:DNA, Plant; D018749:RNA, Plant; D013449:Sulfonamides; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases