Intracellular carbonic anhydrase activities in Dunaliella tertiolecta (Butcher) and Chlamydomonas reinhardtii (Dangeard) in relation to inorganic carbon concentration during growth: further evidence for the existence of two distinct carbonic anhydrases associated with the chloroplasts

Using mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ intracellular carbonic anhydrase (CA) activity was investigated in the unicellular green algae Dunaliella tertiolecta and Chlamydomonas reinhardtii which were either grown on air enriched with 5% CO₂ (high-C_i cells) or on air (low-C_i cells). In D. tertiolecta high- and low-C_i cells had detectable levels of internal CA activity when measured under in-vivo conditions and this activity could be split up into three distinct forms. One CA was not associated with the chloroplasts, while two isozymes were found to be located within the plastids. The activities of all intracellular CAs were always about twofold higher in low than in high-C_i cells of D. tertiolecta and the chloroplastic enzymes were completely induced within 4 h of adaptation to air. One of the chloroplastic CAs was found to be soluble the other was insoluble. In addition to the physical differences, MgSO₄ in vitro caused a more than twofold stimulation of the soluble activity while the insoluble form of CA remained rather unaffected. In C. reinhardtii, MgSO₄ increased the soluble CA activity by 346% and the concentration of MgSO₄ required for half-maximum stimulation was between 10 and 15 mM. Again, the insoluble CA activity was not affected by MgSO₄. Furthermore, the soluble isoenzyme was considerably more sensitive to ethoxyzolamide, a potent inhibitor of CA, than the insoluble enzyme. The concentration of inhibitor causing 50% inhibition of soluble CA activity was 110 and 85 μM ethoxyzolamide for D. tertiolecta and C. reinhardtii, respectively. From these data we conclude that the two chloroplast-associated CAs are distinct enzymes.