Erythrocyte membrane skeleton phosphoproteins: identification of two unrelated phosphoproteins in band 4.9.
Human erythrocyte membrane band 4.9 is phosphorylated by several erythrocyte protein kinases. Chromatography of erythrocyte membrane skeleton proteins on DEAE-Sephacel produces two proteins with relative mobilities, on gel electrophoresis, similar to that of band 4.9. The first, with a molecular mass of 49 kDa, is quite basic (pI greater than 8) while the second, 50.5 kDa, is slightly acidic (pI = 6.2). Comparative two-dimensional peptide mapping reveals that both proteins are present in band 4.9 on one-dimensional gels of total erythrocyte membrane proteins and membrane skeleton proteins. The 49 kDa protein, but not the 50.5 kDa protein, binds to actin filaments in a sedimentation assay. In intact erythrocytes metabolically labeled with [32P]orthophosphate, the 49 kDa protein is phosphorylated by protein kinase C, cAMP-dependent protein kinase, and protein kinases which are active in the absence of exogenous kinase activators. In contrast, the 50.5 kDa protein is phosphorylated by protein kinase C but not by the other protein kinases examined. Finally, two-dimensional peptide mapping was employed to compare the 49 kDa protein and a 57 kDa protein which copurifies with, and has many characteristics of, the 49 kDa protein. Significant similarities were found in both 125I-labeled chymotryptic peptide maps and 32P-labeled tryptic peptide maps, suggesting that the 49 kDa and 57 kDa proteins are closely related.
Horne
W C
WC
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
Miettinen
H
H
Marchesi
V T
VT
D001798:Blood Proteins / Q000032:analysis*; D003598:Cytoskeletal Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D006801:Humans; D007526:Isoelectric Point; D008840:Microfilament Proteins; D010750:Phosphoproteins / Q000097:blood*
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; C044805:DMTN protein, human; D008840:Microfilament Proteins; D010750:Phosphoproteins