Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe.
Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.
D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008301:Maleimides / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism*
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008301:Maleimides; D013049:Spectrin; C010854:N-(1-anilinonaphthalyl)-4-maleimide