Formation of Glycolate by a Reconstituted Spinach Chloroplast Preparation

Abstract

transketolase; 1 j,M U-'4C-fructose-6-P, specific radioactivity 0.4 mcj,'mmole; 20 ,ig of chlorophyll in a total volume of 0.33 ml.The assay was performed at 20 C in a controlled temperature bath equipped with incandescent lighting of 1200 ft-c. The reaction was stopped by adding 0.34 ml of boiling absolute ethyl alcohol, and the tubes were removed to an 80 C water bath for 3 min. The test tubes were then centrifuged at 10,000g for 10 min, and the glycolate present in the supernatant fraction was determined. The supernatant fluid was applied to a Dowex AG 1X8 acetate (100-200 mesh) column (6 X 50 cm), and glycolate was eluted according to the method of Zelitch (33). After application of the sample the column was washed with 15 ml of H20. Glycolate was eluted with 10 ml of 4 M acetic acid. Paper chromatography in a 1-butanol, propionic acid, water solvent (9.4:3:4 v, 'v) confirmed that the eluate contained only glycolate. In this procedure more than 99.9%O of the U-14C-fructose-6-P applied was retained in the column. The recovery of added glycolate-U-14C was better than 95%c A 3-ml aliquot of the acetic acid eluate was placed in 15 ml of a liquid scintillation mixture consisting of 100 g of naphthalene,

Evidence for involvement of 2 types of reaction in glycolate formation during photosynthesis in isolated spinach chloroplasts

“…But the nature of the oxidant itself is still contested: Thus H202 [2,3] generated by a Mehler reaction [4, 51 during photosynthesis or 0, [ 1,6] itself are frequently thought to act as oxidants. Recently Gibbs and coworkers [3,7,8] have concluded mainly from experiments with a reconstituted chloroplast system that 2 oxidation reactions participate in glycolate production, which differ in the nature of the oxidants.…”

Regulation of Soybean Net Photosynthetic CO2 Fixation by the Interaction of CO2, O2, and Ribulose 1,5-Diphosphate Carboxylase

“…This reversible inhibition is associated with the photosynthetic carbon reduction cycle (11), and the inhibition can be reduced by increasing the CO2 concentration, decreasing the 02 concentration, or by reducing the temperature (15,16). Direct inhibition of photosynthetic CO2 fixation has been explained by depletion of Calvin cycle intermediates, particularly the CO6 acceptor RuDP,4 which is presumed to occur because of the 02 stimulation of glycolate synthesis (21,23), or by competitive inhibition of RuDP carboxylase by 02 with respect to CO2 (6,20 (9,21,23,27). This reaction has been shown to occur in the light with chloroplast fragments (23).…”

“…Direct inhibition of photosynthetic CO2 fixation has been explained by depletion of Calvin cycle intermediates, particularly the CO6 acceptor RuDP,4 which is presumed to occur because of the 02 stimulation of glycolate synthesis (21,23), or by competitive inhibition of RuDP carboxylase by 02 with respect to CO2 (6,20 (9,21,23,27). This reaction has been shown to occur in the light with chloroplast fragments (23). Alternatively, Bowes et al (7) showed that RuDP carboxylase can catalyze the oxidation of RuDP by 02 to form P-glycolate.…”

“…(10-12, 19, 20). This reversible inhibition is associated with the photosynthetic carbon reduction cycle (11), and the inhibition can be reduced by increasing the CO2 concentration, decreasing the 02 concentration, or by reducing the temperature (15,16 (9,21,23,27 …”

Production of Glycolate by Oxidation of the 1,2‐Dihydroxyethyl‐thiamin‐diphosphate Intermediate of Transketolase with Hexacyanoferrate(III) or H2O2

“…Oxidation of the 1,2-dihydroxyethyl-thiarnin diphosphate intermediate of transketolase has been proposed as an additional source of glycolate. This hypothesis was deduced from the observation that a preparation of fragmented spinach chloroplasts supplemented with fructose-6-P, transketolase, NADP, and ferredoxin produces glycolate under illumination [4]. In this system, H202 or another unidentified oxidant was thought to oxidize the 'active glycolaldehyde' produced in the transketolase reaction.…”