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Metabolism and Enzymology

Mass Spectrometric Measurement of Intracellular Carbonic Anhydrase Activity in High and Low C_i Cells of Chlamydomonas¹

Studies Using ¹⁸O Exchange with ¹³C/¹⁸O Labeled Bicarbonate

Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6, Department of Biology, University of Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, Federal Republic of Germany

By measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O), intracellular carbonic anhydrase activity was studied with protoplasts and chloroplasts isolated from Chlamydomonas reinhardtii grown either on air (low inorganic carbon [C_i]) or air enriched with 5% CO₂ (high C_i). Intact low C_i protoplasts had a 10-fold higher carbonic anhydrase activity than did high C_i protoplasts. Application of dextran-bound inhibitor and quaternary ammonium sulfanilamide, both known as membrane impermeable inhibitors of carbonic anhydrase, had no influence on the catalysis of ¹⁸O exchange, indicating that cross-contamination with extracellular carbonic anhydrase was not responsible for the observed activity. This intracellular in vivo activity from protoplasts was inhibited by acetazolamide and ethoxyzolamide. Intracellular carbonic anhydrase activity was partly associated with intact chloroplasts isolated from high and low C_i cells, and the latter had a sixfold greater rate of catalysis. The presence of dextran-bound inhibitor had no effect on chloroplast-associated carbonic anhydrase, whereas 150 micromolar ethoxyzolamide caused a 61 to 67% inhibition of activity. These results indicate that chloroplastic carbonic anhydrase was located within the plastid and that it was relatively insensitive to ethoxyzolamide. Carbonic anhydrase activity in crude homogenates of protoplasts and chloroplasts was about six times higher in the low C_i than in high C_i preparations. Further separation into soluble and insoluble fractions together with inhibitor studies revealed that there are at least two different forms of intracellular carbonic anhydrase. One enzyme, which was rather insoluble and relatively insensitive to ethoxyzolamide, is likely an intrachloroplastic carbonic anhydrase. The second carbonic anhydrase, which was soluble and sensitive to ethoxyzolamide, is most probably located in an extrachloroplastic compartment.

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