Radiolabel-transfer cross-linking demonstrates that protein 4.1 binds to the N-terminal region of beta spectrin and to actin in binary interactions.
Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)- succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of beta spectrin within a sequence homologous to the actin-binding region of alpha actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.
Becker
P S
PS
Department of Internal Medicine (Hematology Section), Yale University School of Medicine, New Haven, CT 06510.
Schwartz
M A
MA
Morrow
J S
JS
Lux
S E
SE
D000199:Actins / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000378:metabolism; D003598:Cytoskeletal Proteins; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009479:Neuropeptides; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D013049:Spectrin / Q000302:isolation & purification / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism*; D014357:Trypsin
D000199:Actins; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D013388:Succinimides; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D013049:Spectrin; C047401:1-(N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl)-4-(N-hydroxysuccinimidyl)succinate; D014357:Trypsin