Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes.
A new Mr 43,000 tropomyosin-binding protein (TMBP) has been identified in erythrocyte membranes by binding of 125I-labeled Bolton-Hunter tropomyosin to nitrocellulose blots of membrane proteins separated by sodium dodecyl sulfate-gel electrophoresis. This protein is not actin, because 125I-tropomyosin does not bind to purified actin on blots. Binding of 125I-tropomyosin to this protein is specific because it is inhibited by excess unlabeled tropomyosin but not by F-actin or muscle troponins. This protein has been purified to 95% homogeneity from a 1 M Tris extract of tropomyosin-depleted erythrocyte membranes by DEAE-cellulose and hydroxylapatite chromatography, followed by gel filtration on Ultrogel AcA 44. The purified protein has a Stokes radius of 3.9 nm and a sedimentation coefficient of 2.8 S, corresponding to a native molecular weight of 43,000. Binding of 125I-tropomyosin to the purified TMBP saturates at one tropomyosin molecule (Mr 60,000) to two Mr 43,000 TMBPs, with an affinity of about 5 X 10(-7) M. The TMBP is associated with the membrane skeleton after extraction of membranes with the non-ionic detergent, Triton X-100, and is present with respect to tropomyosin at a ratio of about one for every two tropomyosin molecules. Because there is enough tropomyosin for two tropomyosin molecules to be associated with each of the short actin filaments in the membrane skeleton, the erythrocyte membrane TMBP, together with tropomyosin, could function to restrict the number of spectrin molecules attached to each of the short actin filaments and thus specify the hexagonal symmetry of the spectrin-actin lattice. Alternatively, this TMBP could be homologous to one of the muscle troponins and might function with tropomyosin to regulate erythrocyte actomyosin-ATPase activity and influence erythrocyte shape.
D008841:Actin Cytoskeleton / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D008840:Microfilament Proteins; D008970:Molecular Weight; D051348:Tropomodulin; D014335:Tropomyosin / Q000378:metabolism*
D002352:Carrier Proteins; D008840:Microfilament Proteins; C495829:TMOD1 protein, human; D051348:Tropomodulin; D014335:Tropomyosin