Renal brush border membranes from mice with X-linked hypophosphatemia: protein composition, phosphate binding capacity, and protein kinase activity.
Phosphate uptake by brush-border membrane (BBM) vesicles prepared from hypophosphatemic mice (Hyp) is reduced by half relative to BBM vesicles from normal mice. To investigate this abnormality, we studied the protein composition of BBM, their capacity to bind inorganic phosphate, and their protein kinase activity with and without the addition of exogenous cAMP, in normal and Hyp mice. Gradient polyacrylamide gel electrophoresis of BBM proteins showed 27 bands which were identical in normal and Hyp mice. Incubation of the membranes with ortho[32P]phosphate at 0 degrees C revealed a phosphate binding protein with an apparent molecular weight (Mr) of 79000, which has been previously identified in rats as the monomer of alkaline phosphatase. In normal mice, the Scatchard plot of phosphate binding was not linear, suggesting heterogeneity of the binding sites with two major components. At high substrate concentrations, the affinity (K) was 1.42 mM and maximal binding (Bmax) was 83 pmol/mg protein. At low substrate concentrations, these values were 0.07 mM and 10.9 pmol/mg, respectively. In Hyp mice BBM, only one binding system was found with K and Bmax values of 0.38 mM and 53.8 pmol/mg. Incubation of the membranes with 25 microM[gamma-32P]ATP resulted in the phosphorylation of 11 proteins. The major band (Mr: 79000) corresponded to the inorganic phosphate binding protein, i.e., to the alkaline-phosphatase monomer. The 11 proteins showed maximal phosphorylation at pH 10. The protein of 79000 Mr showed a second peak of phosphorylation at pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007015:Hypophosphatemia, Familial / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*
D010710:Phosphates; D000242:Cyclic AMP; D011494:Protein Kinases; D000469:Alkaline Phosphatase