Co-localization of erythrocyte Ca++-Mg++ ATPase and vitamin D-dependent 28-kDa-calcium binding protein

We have previously shown that the human kidney distal convoluted tubule (DCT) contains epitopes of the human erythrocyte Ca++-Mg++ ATPase pump (J Clin Invest 80: 1225-1231, 1987). To determine whether vitamin D-dependent 28-kilodalton-calcium binding protein (28kDa-CaBP)and Ca++-Mg++ ATPase are present in the same cells of the human kidney, kidney tissue was examined for immunoreactivity with antibodies directed against these proteins. Double-label immunohistochemistry showed that a majority of the distal convoluted tubules contain epitopes to both of these proteins. portions of the distal nephron which were positive for 28kDa-CaBP did not show anti-Ca++-Mg++ ATPase antibody binding. All other portions of the nephron were negative for both proteins. Western blot analysis of kidney homogenates by 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), showed binding of an anti-Ca++-Mg ATPase monoclonal antibody to a major band of Mr = 140,000. Western blots of kidney homogenates by 10% SDS-PAGE also showed binding of an anti-28kDa-CaBP polyclonal antibody to a protein band at Mr = 28,000. Incubation of parallel blots from the same 10% gel with 45CaCl2 demonstrated that the Mr = 28,000 band binds calcium. This work demonstrates, for the first time, that epitopes of vitamin D-inducible 28kDa-CaBp and human erythrocyte Ca++-Mg++ ATPase pump are present in the same cells of the human kidney. Previous work in our laboratory has shown that 28kDa-CaBP binds calcium in a manner analogous to calmodulin, a known regulator of the erythrocyte Ca++-Mg ATPase pump (J Biol Chem, 1987).(ABSTRACT TRUNCATED AT 250 WORDS)