Plasma membrane calcium pump and 28-kDa calcium binding protein in cells of rat kidney distal tubules.
In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Borke
J L
JL
Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Caride
A
A
Verma
A K
AK
Penniston
J T
JT
Kumar
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry / Q000379:methods; D007684:Kidney Tubules / Q000378:metabolism*; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D002147:Calmodulin