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.
Peerce
B E
BE
Department of Physiology and Biophysics, UTMB, Galveston 77555-0641, USA.
Cedilote
M
M
Clarke
R D
RD
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D008871:Microvilli; D009282:Naphthalenesulfonates / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010710:Phosphates / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D050608:Sodium-Phosphate Cotransporter Proteins; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D027981:Symporters; D014364:Tryptophan
D000345:Affinity Labels; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; D010710:Phosphates; D011510:Proteolipids; D050608:Sodium-Phosphate Cotransporter Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D027981:Symporters; C020485:proteoliposomes; C012980:fluorophosphate; C038518:acrylodan; D014364:Tryptophan; D015081:2-Naphthylamine; D005459:Fluorides