ATP-DEPENDENT CARBON TRANSPORT IN PERFUSED CHARA CELLS

Carbon transport across the plasma membrane, and carbon fixation were measured in perfused Chara internodal cells. These parameters were mea sured in external media of pH 5.5 and pH 8.5, where CO2 and HCO3- are, respectively, the predominant carbon species in both light and dark c onditions. Cells perfused with medium containing ATP could utilize bot h CO2 and HCO3- from the external medium in the light. Photosynthetic carbon fixation activity was always higher at pH 5.5 than at pH 8.5. W hen cells were perfused either with medium containing hexokinase and 2 -deoxyglucose to deplete ATP from the cytosol (HK medium) or with medi um containing vanadate, a specific inhibitor of the plasma membrane H-ATPase (V medium), photosynthetic carbon fixation was strongly inhibi ted at both pH 5.5 and 8.5. Perfusion of cells with medium containing pyruvate kinase and phosphoenolpyruvate (PEP) to maximally activate th e H+-ATPase (PK medium), stimulated the photosynthetic carbon fixation activities. Oxygen evolution of isolated chloroplasts and the carbon fixation of cells supplied C-14 intracellularly were not inhibited by perfusion media containing either hexokinase and 2-deoxyglucose or van adate. The results indicate that Chara cells possess CO2 and HCO3- tra nsport systems energized by ATP and sensitive to vanadate in the light . In the dark, intact cells also fix carbon. By contrast, in cells per fused with medium containing ATP, no carbon fixation was detected in 1 mol m-3 total dissolved inorganic carbon (TDIC) at pH 8-5. By increas ing TDIC to 10 mol m-3, dark fixation became detectable, although it w as still lower than that of intact cells at 1 mol m-3 TDIC. Addition o f PEP or PEP and PEP carboxylase to the perfusion media significantly increased the dark-carbon fixation. Perfusion with vanadate had no eff ect on the dark-carbon fixation.