title: Intracellular carbonic anhydrase of Chlamydomonas reinhardtii.
abstract: An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified to homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) lacking a cell wall. Intact cells were washed to remove periplasmic CA and were lysed and fractionated into soluble and membrane fractions by sedimentation. All of the CA activity sedimented with the membrane fraction and was dissociated by treatment with a buffer containing 200 mM KCI. Solubilized proteins were fractionated by ammonium sulfate precipitation, anionic exchange chromatography, and hydrophobic interaction chromatography. The resulting fraction had a specific activity of 1260 Wilbur-Anderson units/mg protein and was inhibited by acetazolamide (50% inhibition concentration, 12 nM). Final purification was accomplished by the specific absorption of the enzyme to a Centricon-10 microconcentrator filter. A single, 29.5-kD polypeptide was eluted from the filter with sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer, and a 1.5 M ammonium sulfate eluate contained CA activity. In comparison with human CA isoenzyme II, the N-terminal and internal amino acid sequences from the 29.5-kD polypeptide were 40% identical with the N-terminal region and 67% identical with an internal conserved region. Based on this evidence, we postulate that the 29.5-kD polypeptide is an internal CA in C. reinhardtii and that the enzyme is closely related to the alpha-type CAs observed in animal species.
mesh_terms: D000086:Acetazolamide / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002473:Cell Wall; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid
chemical_list: D007527:Isoenzymes; D002256:Carbonic Anhydrases; D000086:Acetazolamide