Solubilization and reconstitution of iodide counterflow activity from the thyroid plasma membranes into soybean phospholipid vesicles.
The activity of I- counterflow was solubilized with n-octyl beta-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- counterflow but no apparent Na+-dependent I- transport activity. The activity of I- counterflow was saturated by external I- at the concentration of 50-200 microM, indicating the substrate specificity of the counterflow-mediating activity for I-. The optimal concentration of the detergent for solubilization was near 12.5 mg/ml, as reported in solubilization of some sugar transport carriers of E. coli. The counterflow of I- was inhibited by externally added SCN- but not by ClO4-. 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- counterflow was observed. However, the activity of I- counterflow 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 with N-ethylmaleimide at a concentration of as high as 1 mM did not decrease the activity. In conclusion, it appears that the I- counterflow activity can be solubilized and reconstituted into phospholipid vesicles from the thyroid plasma membranes. The counterflow activity may be susceptible to disulfide-reducing agents.
K
K
Saito
K
K
Yamamoto
K
K
Ando
T
T
Kuzuya
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D066298:In Vitro Techniques; D007454:Iodides / Q000378:metabolism*; D008081:Liposomes; D012995:Solubility; D013025:Glycine max; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism*
D003902:Detergents; D007454:Iodides; D008081:Liposomes; D013439:Sulfhydryl Reagents