Solubilization and Reconstitution of Iodide Counterfiow Activity from the Thyroid Plasma Membranes into Soybean Phospholipid Vesicles

摘要

The activity of I−counterfiow was solubilized withn-octylβ-D-glucopyranoside from the thyroid plasma membranes and reconstituted into phospholipid vesicles made from soybean phospholipids by a detergent dilution procedure. The vesicles exhibited apparent“entrance”I−counterfiow but no apparent Na=-dependent I−transport activity. The activity of I−counterfiow was saturated by external I−at the concentration of 50–200μM, indicating the substrate specificity of the counterflow-mediating activity for I−. The optimal concentration of the detergent for solubiization was near 12.5 mg/ml, as reported in solubilization of some sugar transport carriers ofE. coli. The counterfiow of 1 was inhibited by externally added SCN–but not by CIO4−When solubilized protein was treated with 0.2–1 mM dithiothreitol and with 2–10 mM 2-mercaptoethanol prior to reconstitution, dose-dependent inactivation of I−counterfiow was observed. However, the activity of I−counterfiow was fully preserved when solubilized protein was treated with these agents in the presence of high concentrations of I−(40 and 80 mM). In contrast, treatment withN-ethylmaleimide at a concentration of as high as 1 mm did not decrease the activity. In conclusion, it appears that the I−counterfiow activity can be solubilized and reconstituted into phospholipid vesicles from the thyroid plasma membranes. The counterfiow activity may be susc