Characteristics of Light-Dependent Inorganic Carbon Uptake by Isolated Spinach Chloroplasts

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Abstract
The light-dependent accumulation of radioactively labeled inorganic C in isolated spinach chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated for 60 s at pH 7.6 and 23.degree. C with 0.5 mM sodium bicarbonate, contained 0.5 to 1.0 mM internal inorganic C. The stromal pool of inorganic C increased 5- to 7-fold after 2-3 min of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over 1/2 the inorganic C found in intact spinach chloroplasts after 2 min of light was retained during a subsequent 3-min dark incubation at 5.degree. C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [14C]dimethyloxazolidinedione. About 1/3 of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and 14CO2 bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic C accumulation in the stroma. Bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO2 in the chloroplast.