Oligomeric structure of the sodium-dependent phlorizin binding protein from kidney brush-border membranes.
Immunodetection of solubilized kidney brush-border proteins on Western blots using antibodies against the 70 kDa phlorizin binding component of sodium-glucose cotransporter allows to identify an additional protein band with apparent molecular mass of 120 kDa in the presence of reducing agent dithiothreitol. Antibodies specifically eluted from the 70 kDa protein still recognize the 120 kDa protein on Western blot. The lack of dissociation of the 120 kDa protein from native brush borders or Triton X-100 extract in the presence of dithiothreitol can be improved by an extended incubation at 25 degrees C; this protein is full dissociated when purified by electroelution from polyacrylamide gel and gives two subunits with apparent molecular masses of 70 and 60 kDa by Coomassie staining and Western blot analysis. The effect of dithiothreitol on the renal brush-border membrane phlorizin binding is studied; a decrease in the number of high-affinity phlorizin binding sites without modification of the affinity to the binding molecule is observed. These data suggest that the high-affinity phlorizin binding moiety of sodium-glucose cotransporter exists in the kidney as a dimeric structure.
Gerardi-Laffin
C
C
Laboratoire de Biochimie, Faculté de Médecine, Nice, France.
Delque-Bayer
P
P
Sudaka
P
P
Poiree
J C
JC
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D007672:Kidney Cortex / Q000737:chemistry*; D007687:Kidney Tubules, Proximal / Q000737:chemistry; D008871:Microvilli / Q000737:chemistry; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D012964:Sodium / Q000494:pharmacology; D013552:Swine
D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; C035685:phlorizin-binding protein, mammal; D012964:Sodium; D004229:Dithiothreitol