A 40-kDa polypeptide from papain digestion of the rabbit intestinal Na+/phosphate cotransporter retains Na+ and phosphate cotransport.
The rabbit intestinal brush border membrane Na+/phosphate cotransporter was digested with a variety of proteolytic enzymes. Limited papain digestion generated a 40-kDa polypeptide (P40) which retained putative substrate site markers, fluorescein isothiocyanatophenyl glyoxal and eosin n-acetyl imidazole. P40 retained Na+- and phosphate-selective tryptophan fluorescence quenching, pH sensitivity of ion-induced conformational changes, and tight Na+ and H(2)PO(4)(-) binding. Reconstituted into proteoliposomes, P40 catalyzed Na+-dependent phosphate uptake. The N-terminus of P40 was blocked. An internal sequence of P40 demonstrated that it was derived from NaPi II b. These results suggest that P40 may be a useful model system for studies of the molecular mechanism of Na+-dependent phosphate cotransport and a starting point for structural studies.
Peerce
Brian E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA. bpeerce@utmb.edu
D000595:Amino Acid Sequence; D000818:Animals; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism; D017136:Ion Transport; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010206:Papain; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011510:Proteolipids; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins; D013050:Spectrometry, Fluorescence; D027981:Symporters / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D010446:Peptide Fragments; D010710:Phosphates; D011510:Proteolipids; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C020485:proteoliposomes; D014364:Tryptophan; D012964:Sodium; D010206:Papain