Isolation and characterization of CAB-63, a novel calcium-binding protein.
A novel calcium-binding protein named CAB-63 (formerly called calregulin) has been purified from bovine liver 100,000 X g supernatant. The purified protein has been characterized with respect to its physical, chemical, and calcium-binding properties. It has an apparent molecular weight of 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 55,000 by sedimentation equilibrium centrifugation under nondenaturing conditions. It is an asymmetric molecule with a frictional coefficient of 1.69 and a Stokes radium of 44.2 A. Amino acid analysis has revealed 34.0% acidic residues, 14.0% basic residues, and 4.0% tryptophan. The acidic nature of the molecule is further confirmed by its isoelectric point of 4.65. In the presence of 3 mM MgCl2 and 150 mM KCl, CAB-63 binds 3.0 mol of calcium/mol of protein with an apparent Kd = 0.1 microM. Immunoblotting and Ouchterlony double-diffusion procedures have identified CAB-63 in a variety of bovine tissues. Immunocytochemical staining of both fibroblasts and cryotome-sectioned bovine liver further indicates that CAB-63 immunoreactivity is restricted to an elaborate system of perinuclear membranous vacuoles and cisternae indistinguishable from immunocytochemical staining of the endoplasmic reticulum. It is concluded that CAB-63 represents a major calcium-binding protein whose subcellular organization suggests a possible role in the function of the endoplasmic reticulum.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D037282:Calreticulin; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002848:Chromatography, DEAE-Cellulose; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007526:Isoelectric Point; D008099:Liver / Q000737:chemistry*; D008970:Molecular Weight; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D014018:Tissue Distribution
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D012261:Ribonucleoproteins; D002118:Calcium