Carbonic Anhydrase Activity Associated with the Cyanobacterium Synechococcus PCC7942

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Abstract
Intact cells and crude homogenates of high (1% CO2) and low dissolved inorganic carbon (Ci) (30-50 microliters per liter of CO2) grown Synechococcus PCC7942 have carbonic anhydrase (CA)-like activity, which enables them to catalyze the exchange of 18O from CO2 to H2O. This activity was studied using a mass spectrometer coupled to a cuvette with a membrane inlet system. Intact high and low Ci cells were found to contain CA activity, separated from the medium by a membrane which is preferentially permeable to CO2. This activity is most apparent in the light, where 18O-labeled CO2 species are being taken up by the cells but the effluxing CO2 has lost most of its label to water. In the dark, low Ci cells catalyze the depletion of the 18O enrichment of CO2 and this activity is inhibited by both ethoxyzolamide and 2-(trifluoromethoxy)carbonyl cyanide. This may occur via a common inhibition of the Ci pump and the Ci pump is proposed as a potential site for the exchange of 18O. CA activity was measurable in homogenates of both cell types but was 5- to 10-fold higher in low Ci cells. This was inhibited by ethoxyzolamide with an I50 of 50 to 100 micromolar in both low and high Ci cells. A large proportion of the internal CA activity appears to be pelletable in nature. This pelletability is increased by the presence of Mg2+ in a manner similar to that of ribulose bisphosphate carboxylase-oxygenase activity and chlorophyll (thylakoids) and may be the result of nonspecific aggregation. Separation of crude homogenates on sucrose gradients is consistent with the notion that CA and ribulose bisphosphate carboxylase-oxygenase activity may be associated with the same pelletable fraction. However, we cannot unequivocally establish that CA is located within the carboxysome. The sucrose gradients show the presence of separate soluble and pelletable CA activity. This may be due to the presence of separate forms of the enzyme or may arise from the same pelletable association which is unstable during extraction.