Characterization of 4,4′-Diisothiocyano-2,2′-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O₂ Evolution in Isolated Chloroplasts

Abstract

ABSTRACr 3-Phosphoglycerate (PGA)-dependent 02 evolution by mesophyll chloroplasts of the C4 plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of 02 evolution. The extent of inhibition of 02 evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophylL concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent 02 evolution. Protection inst this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, ifthe chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorgnic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent 02 evolution by isolated chloroplasts of the C3 plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C4 chloroplasts.Oxalacetate-dependent 02 evolution and its stimulation by the uncoupler, NHCICL were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C4 crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.Phosphenolpyruvate is a key regulatory metabolite of the C4 pathway of photosynthesis. It is formed in the mesophyll chlo-

Comparison of the kinetic properties, inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

“…This strongly indicates that all four substrates including PEP are indeed transported by the same translocator protein, as has already been suggested (Day and Hatch 1981;Huber and Edwards 1977b;Rumpho and Edwards 1985). The published kinetic constants (Km 3-phosphoglycerate, Ki phosphate and K~ PEP) for maize mesophyll chloroplasts (Day and Hatch 1981) and Km phosphate and K~ PEP for mesophyll chloroplasts from the C4 plant Digitaria sanguinalis (Huber and Edwards 1977b)) were all substantially higher.…”

“…In C3 plants the export of triose phosphate out of the chloroplasts is catalyzed by a transport protein called the triose phosphate-3-phosphoglycerate-phosphate translocator (in short, phosphate translocator) which transports 3-phosphoglycerate and inorganic phosphate as well (Flfigge and Heldt 1984). Several lines of evidence indicate that, in addition to the transport of the above-mentioned metabolites, the phosphate translocator from C4-mesophyll chloroplasts also catalyzes the transport of PEP (Huber and Edwards 1977b;Day and Hatch 1981;Rumpho and Edwards 1985) although direct evidence for this conclusion is still missing.…”

Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

“…Prerequisite for the operation of C4 photosynthesis is extensive flow of C4 dicarboxylic and C3 acids and triose-P not only intercellularly but also intracellularly (6, 14). Thus, metabolite flow across the envelope of C4 chloroplasts should be different from that of C3 chloroplasts for example, import of pyruvate and export of PEP3 in MC, and export of 3-PGA and import of triose-P in BC, especially in NADP+-malic enzyme type plants such as maize due to the limited availability of reducing power in BC.Experimental studies in MC support some of the expected differentiations in transport systems: existence of a novel translocator in the envelope membrane catalyzing light-dependent pyruvate uptake (12,20) and transport of PEP by PT (4,17,22,23). The PT in the envelope of C3 chloroplasts catalyzing a one to one exchange of Pi, triose-P, and 3-PGA, is now well characterized (7, 10, 11), and its primary structure has been recently determined (8).…”

“…Experimental studies in MC support some of the expected differentiations in transport systems: existence of a novel translocator in the envelope membrane catalyzing light-dependent pyruvate uptake (12,20) and transport of PEP by PT (4, 17,22,23). The PT in the envelope of C3 chloroplasts catalyzing a one to one exchange of Pi, triose-P, and 3-PGA, is now well characterized (7, 10, 11), and its primary structure has been recently determined (8).…”

Inhibition of the Photosynthetic Activities of Isolated Spinach Chloroplasts by Phosphonate Compounds

“…Xylulose 1,5-bisP and carboxyarabinitol bisP are potent inhibitors of rubisco because of their similarity to RuBP (15,19), but are not effective inhibitors with isolated chloroplasts because they cannot readily cross the outer envelope membranes. 4,4-Diisothiocyano-2,2'-disulfonic acid stilbene was recently shown to inhibit the phosphate translocator and PGA reduction by isolated chloroplasts (22). Less specific inhibitors include iodoacetate and iodoacetamide that inhibit many enzymes by reacting with sulfhydryl groups (28) and sulfite which inhibits both rubisco and photophosphorylation (3).…”