Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Abstract

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO2 is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO2 required for the half-maximal rate of photosynthetic O2 evolution (K0.5[CO2]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO2 fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K0.5(CO2) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextranbound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO2 is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO2 from HCO3- at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide, and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO3- transport for internal accumulation might occur at the level of the chloroplast envelope.