Identification of the protein 4.1 binding site to phosphatidylserine vesicles.
Previous studies have shown that protein 4.1 is a multifunctional protein that binds to spectrin, actin, glycophorins, the anion channel protein, and phosphatidylserine (PS). In this report, we have characterized the binding of protein 4.1 and its major proteolytic fragments to phospholipid vesicles. Pure 125I-labeled protein 4.1 was incubated with PS liposomes, and the free protein 4.1 was separated by ultracentrifugation. Protein 4.1 bound to PS liposomes with a high affinity. At saturation, there was 9 X 10(-3) pmol of protein 4.1 bound/pmol of PS with a Kd of 3.3 X 10(-7) M. When the protein 4.1 containing liposomes were examined in an electron microscope, the protein 4.1 was found uniformly decorating the vesicles in a rosettelike fashion. Among peripheral membrane proteins tested (spectrin, actin, ankyrin, and protein 4.1), protein 4.1 showed the highest level of binding to PS. The binding of protein 4.1 to PS, one of the principal phospholipids of the inner half of the lipid bilayer, was considerably higher than the binding to phosphatidylcholine, that is principally located in the outer half of the lipid bilayer. To identify the structural domain of protein 4.1 involved in binding to the phospholipids, a mixture of proteolytic fragments of protein 4.1 was incubated with PS liposomes. The liposomes selectively retained the 30-kilodalton (kDa) basic domain of the protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)
Cohen
A M
AM
Department of Biomedical, St. Elizabeth's Hospital, Tufts University School of Medicine, Boston, Massachusetts 02135.
Liu
S C
SC
Lawler
J
J
Derick
L
L
Palek
J
J
D001798:Blood Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000097:blood*; D009479:Neuropeptides; D010446:Peptide Fragments / Q000032:analysis; D010718:Phosphatidylserines / Q000097:blood*; D011485:Protein Binding
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008081:Liposomes; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D010718:Phosphatidylserines; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1