Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

Abstract

NADP-glyceraldebyde-3-P dehydrogenase of spinach (Spinacis okraces) chloroplasts was activated by thioredoxin that was reduced either photochemicaUy with ferredoxin and ferredoxin-thioredoxin reductase or chemicafly with dithiothreitoL Tle activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis.The NAD-linked glyceradehyde-3-P dehydrogenase activity that Is also present in chloroplasts and in the puried enzyme preparation was not affected by reduced thioredoxin.When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraidehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemicafy reduced thioredoxin was not appreciably affected by oxidizd glutathione. The results suggest that adthough it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraidehyde-3-P debydrogenase differs in its mode of deactivation and in its capacity for actvation by enzyme effectors indpendently of thkoedoxin.The ferredoxin/thioredoxin system is a newly found mechanism that links light to enzyme regulation in chloroplasts (5,6,21,26). In the light, electrons from Chl are transferred to ferredoxin and then via the enzyme ferredoxin-thioredoxin reductase to thioredoxin. Reduced thioredoxin, in turn, activates certain regulatory enzymes of chloroplasts (26,27). In the dark, the activated chloroplast enzymes are deactivated by, depending on the enzyme, either a soluble oxidant (e.g. GSSG,2 dehydroascorbate [261) or an as yet unidentified membrane oxidant (27). In chloroplasts, this, and possibly other (2) "oxidation-reduction" mechanisms may act in conjunction with ion-mediated (9, 24) and effectormediated (14, 18-20, 25) systems of enzyme control.Until now, the only member of the reductive pentose-P cycle shown to be activated photochemically by the defined ferredoxin/thioredoxin system is fructose 1,6-bisphosphatase (6, 26). We now report evidence that another enzyme of the cycle that is known to be activated by light; viz. NADP-glyceraldehyde-3-P dehydrogenase (2, 30) can also be activated by the ferredoxin/thioredoxin system. The regulatory form of NADPglyceraldehyde-3-P dehydrogenase (25) sec, the resultant slurry was filtered through eight layers of filtering silk (Joymar Scientific Co., Hicksville N.Y.), and then centrifuged 1 min at 3,000g. The supernatant fraction was discarded and the green pellet was suspended in 50 mm Tris buffer (pH 7.9) (pH adjusted at 22 C). The concentration of Chl was adjusted to 1.5 mg/ml. The broken chloroplast suspension was centrifuged 10 min at 40,000g. For obtaining chloroplast extract, the supernatant fraction was centrifuged again (1 hr at 105,000g). The precipitate was discarded and the amber supernatant fraction ("chloroplast extract") was used for enzyme assays. For obtaining chloroplast fragments, the pe...

Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype

“…Furthermore, in vitro assays of marker enzymes were carried out to demonstrate that mitochondria (fumarase assay) and peroxisomes (catalase assay) did not contaminate chloroplasts. Glyceraldehyde-3-P dehydrogenase (GAPDH) was used as S-associated chloroplast marker and was assayed in the reductive direction (Wolosiuk and Buchanan, 1976). As shown in Figure 6, GAPDH activity peaked in the S fraction, whereas no activity was detected in the MP.…”

“…Fumarase was assayed by monitoring absorbance of the in vitro-synthesized fumarate at 240 nm according to the method of Racker (1950) with the modification described by Hatch (1978). GAPDH was assayed in the reductive direction as described by Wolosiuk and Buchanan (1976). TDC was assayed according to Pennings et al (1987) with the difference that the in vitro-synthesized tryptamine was detected fluorometrically as described above.…”

Photoactivation of the Water-Oxidation System in Isolated Intact Chloroplasts Prepared from Wheat Leaves Grown under Intermittent Flash Illumination

“…It seems possible to assume in this context that the postulated factor, X, is an SH-bearing substance being reactive with CCCP to lose its function in the photoactivation. It seems also worth while to note that some enzymes for Calvin-Benson cycle, such as NADP-linked glyceraldehyde-3-P dehydrogenase and fructose-1,6-diphosphatase are inactive in darkness but are activated on illumination by a soluble factor in stroma having SH-groups, thioredoxin, reduced via ferredoxin by PSI photoreaction (19,20). It may be reasonable to assume a similar mechanism for the photoactivation of the water-oxidation system, correlating the factor postulated with thioredoxin.…”

“…Actinic light was the red light from an incandescent slide projector (500 w) passing through a red filter (Toshiba, VR-65), a heat absorbing filter (Nihon Shinku, Cold filter-B) and a 10-cm layer of water and focused onto the optical cell at a saturating intensity of 130 mw/cm2. The standard medium for the assay contained 0.33 M sorbitol, 5 mm NaCl, 0.2 mm K2HPO4, 1 mM MgCl2, 20 mm methylamine, 20 /LM DCIP, and 50 mm Hepes (pH 7.5), and 1 mm DPC was added when used. Chl concentration was determined by the method of Arnon (1).…”

Plant Seeds Contain Several Thioredoxins of Regular Size

“…Previously reported properties of plant thioredoxins appeared to support the first view, and the proteins (especially those isolated from leaves) have been grouped into ones activating fructose-bisphosphatase ('f' type), others that preferentially stimulate NADP: malate dehydrogenase ('m' type), and 'c' thioredoxins of presumed cytoplasmic origin [I]. Such typification bears some arbitrariness as other enzymes like ribonucleotide reductases [6, 71, glutamine synthetase [28] or NADP: glyceraldehyde-3-phosphate dehydrogenase [30] are also activated but are not usually measured for practical reasons. In fact, the more or less unlimited exchangeability in v i m of many bacterial.…”