Figure 3.

Stability of soybean root GS polypeptides when the oxidized and nonoxidized forms of the enzyme were incubated in the root extract. A, Four-day-old root extract incubated in the DTT-mediated MCO system in the presence (DTT/Fe·EDTA) or absence of EDTA (DTT/FeCl₃). Samples were taken at the indicated times, and the reaction was terminated by the addition of SDS and boiling. The samples were subjected to SDS-PAGE followed by electroblotting and immunodetection of GS polypeptides using anti-GS antibodies. B, After incubation in the MCO system for 2 h, the extract was passed over a Sephadex G-25 spinout column equilibrated with 50 mm imidazole, pH 7.4, 10 mm DTT, and 1 mm EDTA to remove the Fe³⁺ ion and then incubated for the indicated times. The samples were then subjected to SDS-PAGE followed by electroblotting and immunodetection of GS polypeptides using anti-GS antibodies. C, Bands in A and B were quantified using Intelligent Quantifier software and plotted. ○, DTT/Fe·EDTA treatment; •, DTT/FeCl₃ treatment; ▵, DTT/FeCl₃ system after removal of Fe³⁺. The figure represents typical results.