Effect of Phosphorylated Compounds and Inhibitors on CO₂ Fixation by Intact Spinach Chloroplasts

Abstract

A 2-fold increase in the rate of CO., fixation following the addition of fructose-1,6-diP. ribose-5-P, glucose-1-P, glucose-6-P, fructose-6-P or glyceric acid-3-P to a fragmented spinach chloroplast system was demonstrated by Arnon et al. (1) and Whatley et al. (22). However, there are no reports on the effect of these compounds on the rate of CO., fixation by intact spinach chloroplasts. It became important to extend the work of Arnon.Whatley. and Allen to the intact chloroplast using these and other phosphorylated substances after the demonstration by Havir and Gibbs (14) that the intact chloroplast in sharp contrast to the broken chloroplast preparation possesses the complete reductive pentose-P cycle. In the present work, we report on the varying effects of a number of phosphorylated compounds, principally phosphorylated sugars, upon the uptake of CO., fixation by intact spinach chloroplasts.5 We also report the effects of some of these compounds on CO. fixation in the presence of arsenite and iodoacetamide, 2 substances which inhibit the carbon cycle but appear to have little influence upon the photochemical act (8,11 In experiments where compounds were isolated for isotopic distribution, the system was similar to that described for CO., uptake with the following exceptions: 10 ml of reaction mixture were incubated in 150 ml Warburg vessels and the specific activity of the NaHC140. was increased such that 1 ml of reaction mixture contained 2 moless of NaHCO2 with 50 'Uc of C14. The reaction was stopped by boiling the contents of the flasks for 2 minutes. After centrifugation. an aliquot of the supernatant solution was assayed for isotopic content. Components of the mixture were separated by gradient elution with HCl from a Dowex 1-chloride column (14). Other procedures used were similar to those described in a previous publication (14).Reagents. D-Fructose-1 ,6-diP, D-glucose-6-P. Dribose-5-P, and L-a-glycerol-P were purchased from the Sigma Chemical Company. D-Fructose-6-P was obtained from C. F. Boehringer. D-Erythrose-4-P. D-glyceraldehyde-3-P, dihydroxyacetone-P, glycola.l]dehyde-P, and P-enolpyruvate were purchased from Calbiochem. D-Sedoheptulose-7-P and D-sedoheptulose-1 .7-diP were gifts from B. L. Horecker.

Photosynthetic Production of Hydrogen Peroxide by Anacystis nidulans

“…In fact we were surprised to find effects at all and by such an array of compounds. Depression of the burst by ribose-5-P, ribulose-5-P, fructose-1 , 6-diP, and dihydroxyacetone-P is consistent with observations on shortening of induction effects in chloroplasts (4,5,8,39 We have treated a relatively small production of peroxide observed under special conditions in Anacystis. This observation and our explanation for it are not novel (9,10), save in the sense that we have considered events occurring in intact, unpoisoned cells.…”

“…Recalling effects of various intermediates in decreasing induction lags in 02 production or CO2 uptake by chloroplasts (4,5,8,39), we examined effects of intermediates added at the beginning of the 5-min dark incubation period before assay. Four of the five Calvin cycle intermediates tested depressed H20, production (Table IA).…”

“…1) contained 2 ml of 50 mm phosphate buffer equilibrated with 1% C02, 100 Id of scopoletin stock solution (2 mg/l in water), 1 ml of cell suspension in medium C-m containing 1 Al cells and equilibrated with 1% C02 in air, and 50 Al of a peroxidase solution (4 mg/ml in water). In all experiments, the complete reaction mixture was equilibrated in the cuvette in the dark for 5 min before onset of illumination. The reaction mix was aerated with 1% C02 in air throughout each experiment.…”

Enhanced Dark CO2 Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

“…Alternatively, it may be that the hexose and pentose phosphates formed in the cytoplasm cannot penetrate the photosynthetic apparatus. Studies with the whole chloroplast isolated from higher plants showed that ribose 5-P and fructose 1,6-diP buit not fructose 6-P and glucose 6-P can cross the double memlbrane and enter the photosynthetic carbon reduction cycle (15). An "absoluite" light-dark switch controlling the activitv of riibulose 5-P kinase is unlikelx since at least 2 groups have demonstrated sufficient ribulose 5-P kinase in extracts of photosynthetilc organisms kept in darkness to catalyze the observed rates of CO, fixation (16,17 2-fold higher in illuminated leaves ov-er that fotund in the dark coiitrol (E. Latzko, uinpiublished observations).…”

Studies on the Kinetics of Photosynthetic Products Synthesized by Different Preparations of Intact Spinach Chloroplasts

“…While leaves may have a fixation capacity in the order of 180 ,moles/mg of chlorophyll-hr, the rate obtained by chloroplasts isolated in 0.35 M NaCl was usually less than 5%o of this value (1)(2)(3)6). Then, Walker (11) observed rates as high as 25C-of those of the intact leaf by substituting 0.33 M sorbitol for 0.35 M NaCl in the isolation medium.…”