Carbonic anhydrase activity in isolated chloroplasts of wild-type and high-CO2-dependent mutants of Chlamydomonas reinhardtii as studied by a new assay

In an assay of carbonic anhydrase (CA), NaH14CO3 solution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C reinhardtii had 740 +/- 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 +/- 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 +/- 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw- 15, believed to lack an internal CA, had 2.8 +/- 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 +/- 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 micromolar inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80-C for 10 min inhibited this CA activity by 90.8 +/- 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity