Direct and Indirect Transfer of ATP and ADP across the Chloroplast Envelope

Abstract

1. In intact leaves of Elodea densa illumination resulted in a large increase in the levels of chloroplastic and cytoplasmic ATP and a decrease in chloroplastic and cytoplasmic ADP. Reverse changes were observed on darkening. The kinetics of the fluctuations were similar in chloroplasts and cytoplasm suggesting effective transfer of ATP and ADP between chloroplasts and cytoplasm. Ratios of ATP to ADP were significantly lower in the chloroplasts than in the cytoplasm in the dark and in the light. This may indicate different phosphate potentials in chloroplasts and cytoplasm. Transfer of ATP across the chloroplast envelope as calculated from the light-dependent cytoplasmic ATP increase was 7 to 9 μmoles/mg chlorophyll per hour. Actual transfer rates are probably higher. 2. The determination of the rate of adenylate transfer across the envelope of intact isolated chloroplasts requires information on the composition of chloroplast preparations. Intact chloroplasts were quantitatively separated from envelope-free chloroplasts by density gradient centrifugation on Ludox gradients. The percentage of envelope-free chloroplasts in preparations of intact chloroplasts was also determined from measurements of light-dependent ferricyanide reduction. 3. During isolation in sorbitol buffer ca. 50% of the adenylates were lost from intact chloroplasts which were still capable of high rates of phosphoglycerate reduction and photosynthesis. 4. Adenylate transfer across the envelope of isolated chloroplasts as measured by the light-dependent phosphorylation of added ADP in the absence of cofactors was slow and occured at a rate of 0 to 4 µmoles/mg chloroplyll per hour. In the dark chloroplastic adenylate kinase reacted only very slowly with added AMP and ATP to form ADP. Breakage of the chloroplast envelope stimulated reaction rates. 5. Indirect transfer of ATP and ADP across the chloroplast envelope occurred via a shuttle transfer of phosphoglycerate and dihydroxyacetone phosphate. 3-phosphoglyceraldehyde is not involved as a transport metabolite. Maximum transfer rates of phosphoglycerate and dihydroxyacetone phosphate across the chloroplast envelope were higher than maximum rates of photosynthesis and reached 300 μmoles/mg chlorophyll per hour. Indirect transfer of ATP was somewhat slower than rates of phosphoglycerate reduction by isolated chloroplasts. In vivo transfer of phosphate energy by this transport metabolite system is under control of the redox state of pyridine nucleotides and of the phosphate potential.