The Rate of Photosynthesis in Isolated Chloroplasts

Abstract

Chloroplasts were isolated using aqueous and nonaqueous procedures. Aqueous chloroplasts lost approximately 50 per cent, of their soluble proteins during isolation. Nonaqueous chloroplasts retained all their soluble enzymes, but lost their ability to perform the light reactions of photosynthesis. It was possible to reconstitute a chloroplast system of higher activity by adding soluble enzymes from nonaqueous chloroplasts to protein-deficient aqueous chloroplasts. The properties of the reconstituted chloroplast system were as follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system (∼ 4 μM./. chlorophyll/hr.) was 3–4 times that of the aqueous chloroplasts (∼ I μM./. chlorophyll/hr.). The fixation of aqueous chloroplasts isapparently limited in part by lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast system accumulated PGA. This indicates that the reduction of PGA to triosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 μM. CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to the system, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentrating the reaction mixture. Part of the striking differences of the CO₂-fixing capabilities of chloroplasts in vivo and in vitro is caused by dilution effects. Extrapolation of the dilution effect to the protein concentration which exists in chloroplasts yields a CO₂ fixation rate of approximately 30 μM./mg. chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplasts affect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influence the CO₂ fixation of isolated chloroplasts, it is possible to raise the fixation from approximately 1 per cent, to at least 15 per cent, of the fixation in vivo.