Functional characterization of human erythrocyte spectrin .alpha. and .beta. chains: association with actin and erythrocyte protein 4.1

Abstract

Human erythrocyte spectrin .alpha. and .beta. chains were purified by preparative sodium dodecyl sulfate gel electrophoresis and also by DEAE-cellulose chromatography in the presence of urea. The purified chains behaved as individual monomers on sucrose gradients and did not form homodimers. Recombination of the chains led to the formation of .alpha.-.beta. heterodimers with sedimentation characteristics identical with native .alpha.-.beta. dimers. The binding of 125I-labeled band 4.1 to .alpha. and .beta. chains was measured by sucrose gradient rate zonal sedimentation and by quantitative immunoassay. Both .alpha. and .beta. chains associated with 125I-labeled band 4.1 in a nearly identical manner over the range of band 4.1 concentration studied. The association was abolished by heat denaturation of the spectrin chains or by denaturation of band 4.1 with a 40-fold molar excess of N-ethylmaleimide. As expected, purified .beta. chains but not .alpha. chains bound to 125I-labeled ankyrin as measured by a quantitative radioimmunoassay. The binding of purified .alpha. chains, .beta. chains, and recombinant .alpha.-.beta. heterodimers to F-actin was measured in the presence of band 4.1. .alpha. or .beta. chains separately exhibited no band 4.1 dependent association with F-actin but .alpha.-.beta. heterodimers formed by recombination of the chains did. Spectrin binding to F-actin in the presence of band 4.1 probably requires the participation of both of spectrin''s polypeptide chains.