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

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Abstract
3-Phosphoglycerate (PGA)-dependent O2 evolution by mesophyll chloroplasts of the C4 plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4''-diisothiocyanostilbene-2,2''-disulfonic acid stilbene (DIDS). As little as 1.8 .mu.M DIDS added to the assay medium (containing 0.7 mm PGA) resulted in 80 to 100% inhibition of O2 evolution. The extent of inhibition of O2 evolution observed was dependent on various factors including: pH, concentrations 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 .mu.M levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent O2 evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were 1st incubated with 8.3 mM PGA, phosphoenolpyruvate (PEP) or inorganic PO43- before adding 42 .mu.M 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. .mu.M levels of DIDS also inhibited PGA dependent O2 evolution by isolated chloroplasts of the C3 plant barley. As with crabgrass, preincubation with PGA or inorganic PO43- resulted in a decrease in the DIDS inhibition, but PEP was ineffective compared to the C4 chloroplasts. Oxalacetate-dependent O2 evolution and its stimulation by the uncoupler, NH4Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 .mu.M) 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 PO43- translocator. The data further suggest that in C4 crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.

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