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 ofCO2 required for the half-maximal rate of photosynthetic 02 evolution (Ko4C021) 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 K165(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 HC03-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 HC03-transport for internal accumulation might occur at the level of the chloroplast envelope.

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A Chiamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO2 concentrations but can grow on elevated CO2. In a test cross, the high C02-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO2 accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO2 by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO2 levels to an environment low in CO2. It is concluded that this mutant fails to induce the CO2 concentrating system and is incapable of adapting to low CO2 conditions. Chlamydomonas reinhardtii, like other unicellular green algae, has the capacity to adapt to varying CO2 concentrations in the environment (1-3, 5). When grown on elevated levels of CO2 (5% (v/v) in air), C. reinhardtii has a relatively low affinity for C12 and exhibits high rates of photorespiration when placed in a low CO2/high O2 environment (2,16,19,26). However, if C. reinhardtii remains exposed to low levels of CO2 (ambient CO2 levels), it adapts to these conditions by inducing a CO2 concentrating mechanism (2). This CO2 concentrating mechanism is thought to increase the CO2 at the

An intracellular carbonic anhydrase (CA; EC 4.2.1 .I) was purified to homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) lacking a cell wall. lntact 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. l h e resulting fraction had a specific activity of 1260 WilburAnderson 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. I n 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 i s an internal CA in C. reinhardtii and that the enzyme is closely related to the a-type CAs observed i n animal species.

A physiologically significant level of intracellular carbonic anhydrase has been idenfified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was disfinct from the periplasmic form of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtil periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.The unicellular green alga, Chlamydomonas reinhardtii, has an efficient mechanism for the utilization of Ci3 which allows these cells to carry out photosynthesis at optimal rates when only very low concentrations of CO2 are available in 'Supported in part by the McKnight Foundation for N. E. T., J.V. M., and H.