Adaptive regulation of Na(+)-dependent phosphate transport in the bovine renal epithelial cell line NBL-1. Identification of the phosphate transporter as a 55-kDa glycoprotein.
1. Na(+)-dependent phosphate transport activity from bovine brush border membrane vesicles (BBMV) was reconstituted into artificial phospholipid membranes. It was shown, using lectin affinity chromatography, that transport activity was mediated by a glycoprotein. 2. The bovine kidney epithelial cell line NBL-1 expresses a Na(+)-dependent phosphate transporter which shows adaptive regulation in response to various media phosphate concentrations. Cells incubated in low-phosphate medium and medium containing 10 mM phosphate have Vmax values of 6.34 +/- 0.18 and 2.95 +/- 0.1 nmol.mg-1.3 min-1 and Km values of 17.1 +/- 3.0 and 36.3 +/- 7.14 microM for phosphate transport, respectively. Thus there is a significant (P less than 0.001) decrease in the Km and increase in the Vmax of transport when cells are transferred from 10 mM phosphate medium to low-phosphate medium. 3. Adaptation of phosphate transport in NBL-1 cells to low-phosphate medium was abolished by tunicamycin, an inhibitor of protein glycosylation, indicating that the transporter in these cells, like that in bovine BBMV, is a glycoprotein. 4. Cells were adapted to low-phosphate medium in the presence of [3H]mannose. Under these conditions a single membrane protein of molecular mass 55 kDa was labelled in the absence but not the presence of tunicamycin. Little labelling occurred if the cells were maintained in high-phosphate medium. 5. On the basis of these observations, it is proposed that the phosphate transporter of NBL-1 cells is a 55-kDa glycoprotein. Phosphate is accumulated across the brush border membrane of renal proximal tubule cells via a Na+/phosphate cotransport system. Na(+)-dependent phosphate transport has been extensively studied in isolated brush border membrane vesicles from rabbit and rat kidney [1-3]. The symport of phosphate and Na+ is electroneutral with a stoichiometry of 2Na+/phosphate at physiological pH values.
Department of Biochemistry, University of Bristol, England.
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CR
Helps
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McGivan
D000222:Adaptation, Physiological; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology*; D014415:Tunicamycin / Q000494:pharmacology
D002352:Carrier Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D014415:Tunicamycin; D012964:Sodium; D008358:Mannose