Solubilization and reconstitution of the renal phosphate transporter.
Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.
D000818:Animals; D001149:Arsenates / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D003902:Detergents; D005947:Glucose / Q000378:metabolism; D007668:Kidney / Q000032:analysis*; D008081:Liposomes; D008871:Microvilli / Q000032:analysis; D008970:Molecular Weight; D010206:Papain; D028044:Phosphate-Binding Proteins; D010710:Phosphates / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D012995:Solubility; D013395:Sucrose / Q000494:pharmacology; D014882:Water-Electrolyte Balance
D001149:Arsenates; D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; D028044:Phosphate-Binding Proteins; D010710:Phosphates; D013395:Sucrose; D012964:Sodium; D010206:Papain; D005947:Glucose