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Metabolism and Enzymology

Specific Labeling of the Phosphate Translocator in C₃ and C₄ Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2' -Disulfo-4,4' -Diisothiocyano] Diphenylethane) ¹

Department of Horticulture, The Ohio State University, Columbus, Ohio 43210-1096, Department of Botany, Washington State University, Pullman, Washington 99164, Department of Botany, University of Wisconsin, Madison, Wisconsin 53706

The phosphate translocator protein of C₃ and C₄ mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2' -disulfo-4,4' -diisothiocyano) diphenylethane ([³H]₂-DIDS). Intact mesophyll chloroplasts were isolated from the C₃ plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C₄ plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 µM [³H]₂-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [³H]₂-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C₃ phosphate translocator) for both C₃ and C₄ chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C₃ and C₄ phosphate translocator protein(s).

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