Photosynthetic carbon metabolism in isolated pea chloroplasts: Metabolite levels and enzyme activities

Marques, Ivano A.; Ford, Duane Merlin; Muschinek, Györgyi; Anderson, Louise E.

doi:10.1016/0003-9861(87)90052-x

Cited by 25 publications

(20 citation statements)

References 45 publications

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“…Huber (13) has suggested that glucose 6-P dehydrogenase activity might contribute to the buildup of intermediates during induction. However, in experiments which are comparable to the experiments reported here, we measured metabolite levels and found that glycerate 3-P, triose-P, hexose 1,6-bisP, sedoheptulose 1,7-bisP and ribulose 1,5-bisP levels remained low and apparently constant during the first 2 min of illumination (16). In the present experiments the dehydrogenase was almost completely inactivated during the first 30 s of illumination.…”

supporting

confidence: 89%

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Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Yuan

Anderson²

1987

Plant Physiol.

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Light inactivation of glucose 6-phosphate dehydrogenase is rapid and occurs before photosynthetic 02 evolution is measureable in intact chloroplasts. Likewise, dark activation is rapid. The major light induced change in the kinetic parameters of glucose 6-phosphate dehydrogenase is in maximal velocity.The first enzyme of the oxidative pentose phosphate pathway, glucose 6-P dehydrogenase (EC 1.1.1.49), is inactivated when pea chloroplasts are irradiated (3). This light inactivation probably prevents operation of a futile cycle involving glucose 6-P, NADPH, and oxidative and reductive pentose phosphate pathway enzymes. We have now examined the kinetics of light inactivation of glucose 6-P dehydrogenase in intact chloroplasts during photosynthetic induction and the kinetic parameters of the active (dark) and less active (light) forms of the enzyme.Fickenscher and Scheibe (9) have partially purified cytosolic glucose 6-P dehydrogenase from peas. Unlike the purified chloroplastic enzyme (20), the cytosolic enzyme was not DTT or DTT/thioredoxin sensitive. On the basis ofthis lack of sensitivity to DTT and the results of antibody experiments with extracts from leaves which had been illuminated, Fickenscher and Scheibe concluded that the cytosolic enzyme was not light inactivated. But in their antibody experiments the error in activity determinations for the cytosolic enzyme from darkened and illuminated leaves was 18 and 23%, respectively (Fig. 3 in Ref. 9). Since errors are additive, the error in the determination of light inactivation was then the same as the expected change in activity if the cytosolic enzyme were 40% inactivated by light. Because previous work in this laboratory (4-6) indicated that the cytosolic enzyme was light and DTT inactivated, we have reinvestigated the DTT-dependent inactivation of the enzyme in whole leaf extracts. MATERIALS AND METHODSPhotosynthetic 02 Evolution and Determination of Kinetic Parameters. Chloroplasts were isolated from pea (Pisum sativum L. var Little Marvel) seedlings and photosynthetic 02 evolution assayed as described by Marques and Anderson (15) except that the chloroplast concentration was increased. Where activity with time of illumination was followed, the chloroplast content in the

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confidence: 89%

“…1). Activation of reductive pentose phosphate cycle enzymes occurs at a comparable rate in this system (16). Huber (13) has suggested that glucose 6-P dehydrogenase activity might contribute to the buildup of intermediates during induction.…”

mentioning

confidence: 81%

Changing Activity of Glucose-6-Phosphate Dehydrogenase from Pea Chloroplasts during Photosynthetic Induction

Yuan

Anderson²

1987

Plant Physiol.

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“…In experiments with intact photosynthesizing chloroplasts, we found levels of this sugar to be about 3 mM (15). Inhibition by sedoheptulose-1,7-phosphate2 may then be significant in the intact plant.…”

Section: Characterizationmentioning

confidence: 70%

“…Ribulose-5-phosphate levels in isolated pea chloroplasts are too low to be detected and too low to account for the observed rate of CO2 fixation (15). This observation, the abnormal kinetics seen with the phosphoribulokinase-phosphoriboisomerase pair (6), the physical interaction between phosphoribulokinase and phosphoriboisomerase described here, and the substrate-dependent interaction between yeast phosphoriboisomerase and chloroplastic phosphoribulokinase described previously (22) all suggest that ribulose-5-phosphate may be channeled from the isomerase to the kinase in the chloroplast.…”

Section: Phase Partitioningmentioning

confidence: 96%

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Purification and Characterization of Pea Chloroplastic Phosphoriboisomerase

Skrukrud¹,

Gordon²,

Dorwin³

et al. 1991

Plant Physiol.

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Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.Phosphoriboisomerase (EC 5.3.1.6) participates in the interconversion ofribose-5-phosphate and ribulose-5-phosphate in both the oxidative and reductive pentose phosphate pathways. We have now developed a rapid procedure for the isolation of the chloroplastic phosphoriboisomerase from pea (Pisum sativum) shoots. Although the enzyme has been purified from other sources (24) and spinach ( 18), this is the first time that the enzyme has been purified to apparent homogeneity from the chloroplasts of a higher plant.We have characterized the enzyme with respect to kinetic parameters and inhibitor constants, and we have determined the N-terminal sequence through 34 amino acids. Phase partitioning experiments indicate that the chloroplast phospho-'In memory of Professor Mordhay Avron.

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