Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and (18)O contents of extracellular CO(2) and HCO(3) (-) in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring (18)O exchange between CO(2) and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO(2) hydration rate in different samples of cells that were grown on bubbled air (low-CO(2) conditions). This activity was only weakly inhibited by ethoxzolamide with an I(50) near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO(2)-starved cells there is considerable generation of CO(2) from intracellular stores, a factor that can cause errors in measurement of net CO(2) uptake unless accounted for. It was demonstrated that use of (13)C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO(2) readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO(3) (-) was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO(3) (-) from Synechococcus.