Calcium-induced phosphorylations and [125I]calmodulin binding in renal membrane preparations.
Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from the renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [125I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of 20 microM La2+ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007672:Kidney Cortex / Q000378:metabolism*; D007811:Lanthanum / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D010750:Phosphoproteins; D007811:Lanthanum; D014357:Trypsin; D002118:Calcium