Ca2+-binding protein from human kidney. Purification and properties.
A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 X 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002850:Chromatography, Gel; D006801:Humans; D005779:Immunodiffusion; D007123:Immunoelectrophoresis, Two-Dimensional; D007668:Kidney / Q000032:analysis*; D008970:Molecular Weight; D064030:S100 Calcium Binding Protein G / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium