Renal brush-border-membrane vesicles prepared from newborn rats by free-flow electrophoresis and their proline uptake.
A method for the isolation of brush-border membranes from newborn-rat kidney, employing centrifugation and free-flow electrophoresis, is described. The composition and purity of the preparation was assessed by determination of enzyme activities specific for various cellular membranes. Free-flow electrophoresis resolves the newborn-rat renal membrane suspension into two populations of alkaline phosphatase-enriched brush-border membranes, designated 'A' and 'B', with the A peak also showing activity of (Na+ + K+)-stimulated ATPase, the basolateral membrane marker enzyme, whereas those of the B peak were enriched 11-fold in alkaline phosphatase and substantially decreased in (Na+ + K+)-stimulated ATPase activity. Membranes in the A peak showed a 7-fold enrichment of alkaline phosphatase, and (Na+ + K+)-stimulated ATPase activity similar to that of the original homogenate. Proline uptake employed to assess osmotic dependency revealed 7% binding of proline to the B vesicles and 31% to the A vesicles. This contrasts with 60% proline binding to vesicles prepared by centrifugation alone. Unlike vesicles from adult animals, proline uptake by B vesicles did not show an Na+-stimulated overshoot, but did exhibit an Na+-gradient enhanced rate of early proline entry. proline entry.
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D004586:Electrophoresis; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D009994:Osmolar Concentration; D011392:Proline / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D011392:Proline