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NaCl, KCl, and sucrose at equiosmolar concentrations had similar inhibitory effects on photosynthetic carbon metabolism by the freshwater green alga, Chlamydomonas reinhardtii. Inhibitory concentrations of these solutes altered the products of photosynthetic (14)CO(2) incorporation, resulting in reduced incorporation into starch, sugar phosphates, lactate, and glycolate, but caused an accumulation of glycerol both intracellularly and in the medium.

The levels of activity of 2-phosphoglycolate phosphatase in the green algae, Chlamydomonas reinhardtii and Chlorella vulgaris, were in the range of 37 to 60 micromoles per milligram chlorophyll per hour and in the blue-green algae, Anacystis nidulans and Anabaena variabilis were 204 to 310 micromoles per milligram chlorophyll per hour. The activity in each species was similar regardless of whether the algae were grown with air or 5% CO(2) in air. The enzyme purified 530-fold from Chlamydomonas was stable, had a broad pH optimum between 6 and 8.5, and was specific for the hydrolysis of P-glycolate with a K(m) of 23 micromolar. The enzyme purified 18-fold from Anacystis was labile, had a sharp pH optimum at 6.3, and was also specific for P-glycolate with a K(m) of 94 micromolar. The molecular weight of the enzyme from Chlamydomonas was estimated to be 92,000 by gel filtration.The phosphatase from both sources required a divalent cation for activity. The Chlamydomonas enzyme was most effectively activated by Co(2+), but was also activated by Mg(2+) (K(a) = 30 micromolar), Mn(2+), and Zn(2+). The Anacystis enzyme was most effectively activated by Mg(2+) (K(a) = 140 micromolar), and was also activated by Co(2+) and Mn(2+), but not by Zn(2+). Anions were also required for maximum activity of the enzyme from both sources. The Chlamydomonas enzyme was activated about 2- to 3-fold by chloride (K(a) = 140 micromolar), bromide, nitrate, bicarbonate (K(a) = 600 micromolar) and formate. The Anacystis enzyme was activated over 10-fold by chloride (K(a) = 870 micromolar), bromide, iodide, and nitrate, but was not activated by bicarbonate or formate.The properties of the algal enzymes were similar to those previously reported for higher plants. The levels and kinetic properties of the enzyme seemed sufficient to account for the flux through the glycolate pathway that occurs in these algae. The phosphatase was not associated with the ribulose 1,5-bisphosphate carboxylase/oxygenase responsible for P-glycolate formation in the carboxysomes of Anacystis.

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 CO(2) is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO(2) required for the half-maximal rate of photosynthetic O(2) evolution (K(0.5)[CO(2)]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO(2) fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K(0.5)(CO(2)) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO(2) is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO(2) from HCO(3) (-) 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 HCO(3) (-) transport for internal accumulation might occur at the level of the chloroplast envelope.

A Chlamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO(2) concentrations but can grow on elevated CO(2). In a test cross, the high CO(2)-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 CO(2) 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 CO(2) 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 CO(2) levels to an environment low in CO(2). It is concluded that this mutant fails to induce the CO(2) concentrating system and is incapable of adapting to low CO(2) conditions.