The inward and outward fluxes of inorganic carbon in illuminated cell suspensions of air-grown Anabaena variabilis were measured by mass spectrometry under conditions of inorganic carbon disequilibrium. The inclusion of 25 mM NaCl significantly enhanced both inward inorganic carbon influx during CO2 fixation and outward CO2 efflux when CO2 fixation was blocked by the Calvin cycle inhibitor, iodoacetamide. At low, steady-state concentrations of inorganic carbon (< 100μM), CO2 fixation was nearly entirely supported by HCO3− transport in the presence of 25 mM NaCl. At approximately 150 μM inorganic carbon, the contributions of CO2 and HCO3− transport to CO2 fixation were about equal. Above this, CO2 transport provided most of the substrate for CO2 fixation. The affinity (K0.5) of photosynthesizing cells for CO2, HCO3− and total inorganic carbon was determined and mean values of 1.7, 9.5, and 8.2 μM, respectively, were determined. Maximum rates of inward CO2 and HCO3− transport and CO2 fixation during steady state were 255.7, 307.3, and 329.1 μmol∙mg−1 Chl∙h−1, respectively. Permeability coefficients for CO2 of 9.8 × 10−8 m∙s−1 and 2.8 × 10−7 m∙s−1 were calculated for the plasma membrane and carboxysomal surface areas, respectively, from the dark efflux rates assuming an internal pH of 7.2. A permeability coefficient for HCO3− across the plasma membrane of 7.6 × 10−9 m∙s−1 was calculated from the dark inorganic carbon efflux corrected for the corresponding dark CO2 efflux. Sodium sulphide (Na2S, 200 μM) blocked CO2 transport. In the presence of 25 mM NaCl, net CO2 efflux was approximately seven times greater than in its absence, when CO2 transport and fixation were both blocked, indicating greater CO2 leakage as a result of larger internal inorganic carbon pools in the presence of NaCl. The rapidity and amount of C16O2 generated from the exchange of 18O from 18O-enriched HCO3− with water in cell suspensions suggested that the internal inorganic carbon pool may be rapidly equilibrated. Key words: Anabaena variabilis, CO2-concentrating mechanism, CO2 transport, HCO3− transport, CO2 efflux, permeability coefficient.
Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane
