Examination of the molecular mechanism of SH reagent-induced inhibition of the intestinal brush-border membrane Na+/phosphate cotransporter.

SH residues on the rabbit intestinal brush-border membrane Na+/phosphate cotransporter were examined using a variety of SH specific reagents, proteolytic digestion and HPLC separation of SH-labeled cotransporter, and partial reaction assays. Of the seven SH-containing peptide fragments on the non-denatured non-reduced cotransporter six peptides were labeled: five SH-containing peptides were labeled with acrylodan or IAF (iodoacetamidofluorescein) and three peptides were labeled with IAEDANS. One SH-containing peptide was labeled with IAEDANS or fluorescein maleimide only. Selective SH labeling conditions employing acrylodan and IAEDANS were used to identify the environments of these SH-containing peptides in the native cotransporter. The nature of SH reagent-induced inhibition of Na(+)-dependent phosphate uptake was examined using substrate-induced conformational changes, and substrate-induced changes in IAEDANS and acrylodan fluorescence of the SH-labeled Na+/phosphate cotransporter. The results indicate that five of the SH-labeled peptides sense the Na(+)-induced conformational change, three peptides sense the Na++ difluorophosphate-induced conformational change, and one peptide senses only the Na++ monofluorophosphate-induced conformational change. Five of the SH-labeled peptides are passive participants in the substrate-induced conformational changes with only SH 51 involved in cotransporter function. Alkylation of SH 51 resulted in a cotransporter conformation which differed from the substrate-mediated conformations and was characterized by increased monofluorophosphate sensitivity.