A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification.
A new protein that binds calmodulin has been identified and purified to greater than 95% homogeneity from the Triton X-100-insoluble residue of human erythrocyte ghost membranes (cytoskeletons) by DEAE chromatography and preparative rate zonal sucrose gradient sedimentation. This ghost calmodulin-binding protein is an alpha/beta heterodimer with subunits of Mr = 103,000 (alpha) and 97,000 (beta). The protein exhibits a Stokes radius of 6.9 nm and a sedimentation coefficient of 6.8 S, corresponding to a molecular weight of 197,000. Moreover, the protein is cross-linked by Cu2+/phenanthroline to a dimer of Mr = 200,000. The Mr = 97,000 beta subunit was identified as the calmodulin-binding site by photoaffinity labeling with 125I-azidocalmodulin. A 230 nM affinity for calmodulin was estimated by displacement of two different concentrations of the 125I-azidocalmodulin with unmodified calmodulin and subsequent Dixon plot analysis. This calmodulin-binding protein is present in erythrocytes at 30,000 copies/cell and is associated exclusively with the membrane. It is tightly bound to a site on red cell cytoskeletons and is totally solubilized in the low ionic strength extract derived from red cell ghost membranes. Visualization of this calmodulin-binding protein in the electron microscope by rotary shadowing, negative staining, and unidirectional shadowing indicates that it is a flattened circular molecule with a 12.4-nm diameter and a 5.4-nm height. Affinity-purified antibodies against the calmodulin-binding protein identify a cross-reacting Mr = 100,000 polypeptide(s) in brain membranes.
D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004910:Erythrocyte Membrane / Q000032:analysis*; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D007163:Immunosorbent Techniques; D008433:Mathematics; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008854:Microscopy, Electron; D008970:Molecular Weight; D010618:Phenanthrolines / Q000378:metabolism; D012995:Solubility; D000099291:Adducins
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007074:Immunoglobulin G; D008565:Membrane Proteins; D010618:Phenanthrolines; D000099291:Adducins; C025205:1,10-phenanthroline