Chromatofocussing and centrifugal reconstitution as tools for the separation and characterization of the Na+-cotransport systems of the brush-border membrane.
Chromatofocussing was used for the separation of brush-border membrane proteins from calf kidney into 4 or 5 fractions over the pH range 4.0 to 7.4. These fractions were reconstituted into proteoliposomes by gradient centrifugation. Determination of the sodium-dependent solute uptake by proteoliposomes reconstituted from different chromatofocussed fractions showed that the sodium-D-glucose cotransport system was present in the fraction eluted between pH 5.3 and 5.8, and that the sodium-phosphate cotransport was present in fractions eluted between pH 4.6 and 5.3 and between pH 5.8 and 6.6, sodium-alanine cotransport could be detected in almost all fractions. Marker enzymes of the brush-border membrane, such as alkaline phosphatase, gamma-glutamyltransferase and aminopeptidase M etc. were also found to be eluted at pH 7.0-7.4, 4.0-4.1 and 5.6-5.8, respectively. These results suggest that chromatofocussing is a promising tool for the separation of membrane proteins and for pre-purification of the sodium-D-glucose cotransport system. It can be further concluded that the sodium-dependent phosphate transport across the brush-border membrane is not dependent upon alkaline phosphatase activity.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007672:Kidney Cortex / Q000032:analysis / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000032:analysis; D008871:Microvilli / Q000032:analysis; D009004:Monosaccharide Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins