A molecular defect in two families with hemolytic poikilocytic anemia: reduction of high affinity membrane binding sites for ankyrin.
Patients from two families with chronic hemolytic anemia have been studied. The erythrocytes are very fragile and appear microcytic with a great variety of shapes. Clinical evaluation failed to identify traditionally recognized causes of hemolysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed no significant abnormality of the major polypeptide bands. Erythrocytes spectrin-ankyrin and ankyrin-membrane interactions were analyzed with 125I-labeled spectrin, 125I-labeled ankyrin, and inside-out vesicles. Patients' vesicles bound 125I-spectrin normally. Likewise, patients' spectrin and ankyrin competed normally for the binding sites on control membranes. None of the individual components appeared to have abnormal thermal sensitivity. Ankyrin-stripped, inside-out vesicles prepared from the patients bound less 125I-ankyrin than did vesicles prepared from normals (P less than 0.05 for all corresponding points in the high-affinity region). Scatchard analysis showed the most significant abnormality to be a 50% reduction in the high affinity ankyrin binding sites. Similar experiments were performed with blood from patients with spherocytosis and splenectomized controls, but no abnormalities were detected. The water soluble 43,000-dalton fragments of band 3 (the high-affinity ankyrin binding sites) were prepared from one of the patients and competed normally for 125I-ankyrin binding in solution. This suggests that the primary structural defect is a reduction in the number of high affinity membrane binding sites for ankyrin, and is consistent with an abnormal organization of band 3 in the membrane.
D000328:Adult; D000368:Aged; D000743:Anemia, Hemolytic / Q000097:blood*; D017487:Ankyrins; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004913:Erythrocytes, Abnormal / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D010455:Peptides / Q000032:analysis; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism; D013156:Splenectomy
D017487:Ankyrins; D008565:Membrane Proteins; D010455:Peptides; D013049:Spectrin