Publications [#112954] of G Vann V. Bennett
- K Gardner, V Bennett, A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification.,
The Journal of biological chemistry, UNITED STATES, vol. 261 no. 3
pp. 1339-48, ISSN 0021-9258
(last updated on 2003/02/18)
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.
Calmodulin • Calmodulin-Binding Proteins • Chromatography, Affinity • Chromatography, Ion Exchange • Erythrocyte Membrane • Humans • Immunoglobulin G • Immunosorbent Techniques • Mathematics • Membrane Proteins • Microscopy, Electron • Molecular Weight • Phenanthrolines • Solubility • analysis* • isolation & purification • isolation & purification* • metabolism • metabolism*