Reconstitution of the partially purified renal phosphate (Pi) transporter.
Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.
D000818:Animals; D001149:Arsenates / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000494:pharmacology
D001149:Arsenates; D002352:Carrier Proteins; D008081:Liposomes; D008565:Membrane Proteins; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D011510:Proteolipids; C036541:phosphate-binding proteolipid; D012964:Sodium; C025657:arsenic acid