Examination of the Na+-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes.
The Na+-induced change in conformation of the intestinal brush border glucose carrier has been examined by three procedures. In the first, we have measured the effect of Na+ on the binding of fluorescein isothiocyanate (FITC) to the glucose site; 100 mM Na increased the specific [blocked by D-glucose, p-(chloromercuri)benzenesulfonic acid, and N-acetylimidazole] FITC binding to a 75-kilodalton polypeptide 3-fold. In the second series, we have examined the effect of Na+ on the susceptibility of the fluorescently labeled glucose site [pyrene isothiocyanate (PYTC) labeled] to a hydrophilic quencher (Tl+); 100 mM NaCl increased the fraction of PYTC sites available to Tl+ from 32% to 92% and decreased the apparent quenching constant from 94 to 44 M-1. Finally, in the third series, we probed the distribution of tryptophan residues 15-30 A from the glucose site using a "distant reporter group method", where tryptophan was used an an energy donor to anthracene isothiocyanate bound to the glucose site. Tryptophan quench reagents (I-, Cs+, and acrylamide) were then employed to probe the accessibility of the glucose site tryptophans in the presence and absence of sodium. In the absence of Na+, there were two major classes of glucose tryptophans--exterior surface residues and residues buried in the hydrophobic protein matrix. Na+ caused a redistribution of the donor tryptophans such that a higher percentage were accessible to I- (51% vs. 25%) and fewer were accessible to Cs+ (13% vs. 25%) and acrylamide (27% vs. 57%).(ABSTRACT TRUNCATED AT 250 WORDS)
Peerce
B E
BE
Department of Physiology, School of Medicine, University of California, Los Angeles 90024.
Wright
E M
EM
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D005456:Fluorescent Dyes; D005947:Glucose / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D017879:Isothiocyanates; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology*; D013691:Tellurium / Q000494:pharmacology; D013861:Thiocyanates; D014364:Tryptophan
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D013861:Thiocyanates; C037152:isothiocyanic acid; D014364:Tryptophan; D012964:Sodium; D005947:Glucose; D013691:Tellurium