Accumulation of bicarbonate in intact chloroplasts following a pH gradient

Abstract

With silicone layer filtering centrifugation the uptake of radioactively labelled bicarbonate into isolated spinach chloroplasts was followed. This uptake was shown to have the following properties: 1. (a) It is so rapid that the kinetics of uptake usually cannot be resolved. 2. (b) Bicarbonate is accumulated in the stroma. The factor between the internal and external concentrations increases greatly when the pH of the medium is lowered from pH 8.5 to pH 7.0. 3. (c) The accumulation factor is independent of the concentration in the medium for a long concentration range. 4. (d) The accumulation of bicarbonate is increased when the chloroplasts are illuminated. This increase is abolished by the addition of uncoupler. 5. (e) Diamox, an inhibitor of carbonic anhydrase, inhibits the rate of bicarbonate uptake. The activity of carbonic anhydrase was assayed in isolated chloroplasts and in leaf homogenates. In agreement with earlier reports the main activity was found to be located in the chloroplasts. This activity is latent; it can be only assayed if the chloroplasts are osmotically shocked. From these results the following conclusions have been drawn: 1. (a) The inner membrane is impermeable to protons. Light-driven proton transport into the thylakoid space causes an alkalisation of the stroma. 2. (b) The uptake of bicarbonate proceeds via diffusion of CO 2 across the inner membrane. There are no indications for a specific transport of bicarbonate. 3. (c) The CO 2 concentration in the chloroplasts may be equal to the CO 2 concentration in the external space. The distribution of bicarbonate between the two compartments is inversely proportional to the distribution of protons. A possible involvement of carbonic anhydrase and the bicarbonate pool in the stroma in increasing the CO 2 affinity of CO 2 fixation is discussed.