Stimulation of Carbon Dioxide Fixation in Isolated Pea Chloroplasts by Catalytic Amounts of Adenine Nucleotides

Abstract

Carbon dioxide-dependent 02 evolution by isolated pea (Pisum sativum var. Massey Gem) chloroplasts was increased two to 12 times by the addition of ATP. 02 evolution was also stimulated by ADP and to a lesser extent by AMP. The ATP effects were not due to broken chloroplasts present in the preparations nor was ATP acting as a phosphate source. We concluded that the adenine nucleotides were acting catalytically. The concentration of ATP required for half-maximum rate of 02 evolution was 16 to 25 FAM. The degree to which ATP stimulated 02 evolution depended on the age of pea plants from which the chloroplasts were isolated. Spinach (Spinacia oleracea var. True Hybrid 102) chloroplasts did not show a consistent stimulation of 02 evolution by adenine nucleotides.The adenine nucleotide content of pea chloroplasts was not lower than that of spinach chloroplasts, but pea chloroplasts which showed a large stimulation of 02 evolution by ATP contained an ATP-hydrolyzing reaction with rates of 10 to 50 iLmol ATP hydrolyzed mg chlorophyll-' hour-'. The rate of the ATP-consuming reaction was much lower in spinach chloroplasts and in chloroplasts from older pea plants which did not show large stimulation of 02 evolution by ATP. We propose that the ATP-consuming reaction, with a high affinity for ATP, decreased the effective size of the ATP pool available for CO2 frixation. Added adenine nucleotides could be transported into the chloroplasts increasing the concentration of internal nucleotides. Calculations showed that the adenine nucleotide transporter on the outer chloroplast membranes could operate at a sufficient rate to produce such an effect. Adelaide 5001, Australia tion of 02 evolution by uncoupling agents. These reports suggest that adenine nucleotides penetrate intact chloroplasts to some extent. Jensen and Bassham (11) showed that ATP stimulated CO2 fixation to a small extent in intact chloroplasts, and that in the dark, ATP increased the level of ribulose-1,5-diphosphate. Studies of diffusion of compounds from isolated chloroplasts suggested that ATP and ADP diffuse out of chloroplasts but can re-enter and be used metabolically (2). Heldt (9) reported the presence of an adenine nucleotide transporter on the chloroplast outer membrane which apparently operates primarily to move ATP into the chloroplast, although the rates of transfer were low (5 ,umol mg Chl-' hr-'). The experiments of Heber and Santarius (8) similarly suggested low rates of adenine nucleotide transport into chloroplasts. Stokes and Walker (22) suggested that the failure of ATP to reverse uncoupler inhibition of P-glyceratedependent 02 evolution indicated impermeability of the chloroplast outer membrane to adenine nucleotides. It should be noted, however, that the adenine nucleotide transporter is inhibited by uncouplers (9), and that reversal of uncoupler inhibition requires the accumulation of substrate quantities of ATP.Stimulation of C02-dependent 02 evolution by ATP does not necessarily imply entry of ATP into chloroplasts. The ATP coul...

Polarographic Study of Ammonia Assimilation by Isolated Chloroplasts

“…The results in Figure 4 emphasize that 2 mm ammonia does not cause any appreciable inhibition of a metabolic activity dependent on both photophosphorylation and electron transport in intact chloroplasts. However, 8 mm ammonia caused 57% inhibition of C02-dependent 02 evolution by pea chloroplasts (22) and this value is in close agreement with that described for (ammonia plus a-ketoglutarate)-dependent activity (Fig. 4, 56% inhibition at 8 mM).…”

“…We suspect that they are required for reaction I (4, l) 22) and that a favorable concentration of MgCl2 is also required for reaction 11 (20). While Mitchell and Stocking (18) reported that chloroplasts catalyzed glutamine synthesis in the absence of ADP, Robinson and Wiskich (22) found that chloroplasts from young peas required catalytic amounts of ADP or ATP to /b 6 promote maximum rates of CO2-dependent 02 evolution whereas those from more mature peas did not. PPi has been used as a slow source of Pi for CO2 fixation by isolated chloroplasts (4).…”

“…This value is in good agreement with the N flux of glutamine formation by glutamine synthetase (18) and glutamine metabolism by glutamate synthase (1) catalyzed by isolated pea chloroplasts of similar intactness to those used in the present study. (22); this represents an average photosynthetically' coupled C flux of 100 jig atoms mg Chl-' hr-t. Isolated pea chloroplasts therefore have relative photosynthetically coupled C to N fluxes of 6:1 (atom/atom) whereas typical C to N ratios of plant material are 40:1 (atom/atom) (6). These considerations show that chloroplasts have the capacity to assimilate more ammonia relative to CO. than required to produce the typical C to N ratios of plant material.…”

Photosynthetic protein phosphorylation in intact chloroplasts

“…NH4CI (5 mM), catalase (5000 units) and D,L-glyceraldehyde (10 mM) were also present where indicated. The inclusion of Na4P207 and ATP was found to be necessary in order to obtain appreciable rates of CO*-dependent 0, evolution [6,7]. The total volume of each reaction mixture was 2.0 ml (5.0 ml for fig.3) and chlorophyll (chl) was 50 pg/ml.…”

Quantitative Local Photosynthetic Flux Measurements at Isolated Chloroplasts and Thylakoid Membranes Using Scanning Electrochemical Microscopy (SECM)

“…Oxygen evolution in the presence of ferricyanide is a well established method for measuring the proportion of burst chloroplasts in an ensemble in suspension. [48][49][50][51] Ferricyanide is a well-known anionic, non-physiological electron acceptor (producing ferrocyanide) which can only freely interact with thylakoid membranes upon bursting of the chloroplast envelope. 52 Once through the chloroplast envelope, the impermeability of the thylakoid membrane only allows the ferricyanide/ferrocyanide redox couple to act on the stromal side of photosystem II (PSII).…”