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)
Borke
J L
JL
Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Minami
J
J
Verma
A K
AK
Penniston
J T
JT
Kumar
R
R
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis*; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D000939:Epitopes / Q000032:analysis; D004910:Erythrocyte Membrane / Q000032:analysis*; D006801:Humans; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D007684:Kidney Tubules / Q000032:analysis*; D007686:Kidney Tubules, Distal / Q000032:analysis*; D064030:S100 Calcium Binding Protein G / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D064030:S100 Calcium Binding Protein G; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases