Specific Labeling of the Phosphate Translocator in C3 and C4 Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (12-Ditritio-12-22′ -Disulfo-44′ -Diisothiocyano Diphenylethane)

摘要

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).