Human Erythrocyte Spectrin Research Articles

The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes

Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10--15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.

The molecular structure of human erythrocyte spectrin: Biophysical and electron microscopic studies

Molecules of human erythrocyte spectrin have been examined by electron microscopy after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramere were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the two polypeptide chains, band 1 (240,000 M r) and band 2 (220,000 M r), often appeared as two separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appears to be weak, except at both ends of the molecule where there are sites of strong binding. Tetramers are formed by the end-to-end association of two spectrin heterodimer molecules without measurable overlap, and have a mean shadowed length of 194 nm. This association to form tetramers probably involves head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode are not observed. The molecular shape of spectrin is quite distinct from that of myosin, to which it has often been likened.

Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin

Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin

Specific fragmentation of human erythrocyte spectrin by chemical cleavage at cysteine residues

Spectrin, isolated from human erythrocyte membrane, was specifically cleaved at the amino side of its cysteine residues by reacting it with 2-nitro-5-thiocyanobenzoic acid at pH 8.0 and incubating the product at pH 9.0. Conditions were developed to obtain quantitative cleavage, with virtually no side reactions due to exposure to the alkaline pH. The solubility and aggregation state of the spectrin fragments in 0.2 M sodium chloride, in 7 M guanidine hydrochloride or in 10 M urea, at pH 8.0, allow separation and partial purification of the fragments by gel filtration or by ion-exchange chromatography. Our results strongly suggest that various parts of the spectrin molecules have similar amino acid compositions. Due to the relatively limited number of fragments, this cleavage method is a promising tool for further elucidation of the structure of spectrin and for understanding its role in the erythrocyte membrane.

Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin.

Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin.

Phospholipid composition of human erythrocyte spectrin.

Human erythrocyte spectrin, which has been extracted at low ionic strength and precipitated at pH 5.1, contains 0.025 mumoles phospholipid/mg protein. The composition of the spectrin-phospholipids differs from that of the erythrocyte membrane. A remarkable similarity was found between the composition of the "available" phospholipids of the inner leaflet of the membrane bilayer and the spectrin-associated phospholipids. Rebinding studies with 125I-labeled spectrin show that the labeled spectrin binds preferentially to the cytoplasmic side of the membrane and that this binding is influenced by protein perturbations.

Human erythrocyte spectrin: Phosphorylation in intact cells and purification of the 32P-labeled protein in a non-aggregated state

[ 32P]spectrin (0.5 Ci/mMole) has been isolated from human erythrocytes incubated with 32Pi and purified to homogeneity by preparative rate zonal sedimentation on linear sucrose gradients. 32P-label, localized in band 2, co-elutes with spectrin from ghosts with a similar dependence on ionic strength and Mg ++ ion, and has the same sedimentation coefficient and an identical effective Stokes radius. [ 32P]spectrin reassociates in a specific manner with spectrin-depleted membranes. Bands 1 and 2 bind in equal ratios, and the 32P-label is distributed with band 2. Purified [ 32P]spectrin is not aggregated since this protein migrates as a symmetrical peak on Sepharose(C1)4B at about 1.6 Vo and sediments at 8S 20,w on sucrose gradients.

Crosslinking studies with different length dithiobisalkylimidates (I) solubilized erythrocyte spectrin

A series of different length dithiobisalkylimidate homologues was synthesized via Bunte salts from corresponding ω-chloronitriles. And by using these reagents, crosslinking of human erythrocyte spectrin was examined. Shorter reagents formed three bands in the region expected for a dimer, all of which were identified as the bands 1–2 associate: bands 1 and 2 polypeptides were crosslinked only each to one another and not to itself. Longer reagents, however, produced higher multimers, which was enhanced by higher temperatures and divalent cations, and depressed by Triton X-100 and deoxycholate. The results suggest the validity of the chain length of the reagents in discriminating between dimer and multimer of spectrin.

Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells.

Important similarities are reported between human smooth muscle actomyosin and the human erythrocyte spectrin complex, primarily components 1, 2, and 5 (Fairbanks G., Steck, T.L., and Wallach, D.F.H. (1971), Biochemistry 10, 2606). The actin-like protein, component 5, is identical with human uterine actin in its ability to form 50-70-A filaments to stimulate myosin ATPase activity, and to bind rabbit heavy meromyoson specit heavy meromyosin specifically. Antibodies to human smooth muscle myosin(uterine) were prepared which were monospecific. A weak but specific cross-reaction of these antisera with components 1 and/or 2 (spectrin) was characterized and at least 25% of the antimyosin antibodies showed a low affinity reaction iwth spectrin. Antibodies generated against a soluble complex of spectrin components 1 and 2 reacted only with component 1 and did not cross-react with myosin. In addition to these structural similarities between smooth muscle actomyosin and the spectrin complex, we have found that spectrin is involved in ATP-dependent erythrocyte shape changes (Sheetz, M.P., Painter, R.G., AND Singer, S.J. (1976B), Cold Spring Harbor Symp. Cell Motility (in press) and, therefore, the spectrin complex is also a mechanochemical protein system.

Extraction and localization of a (Ca 2+ + Mg 2+)-stimulated ATPase in human erythrocyte spectrin

1. (1) A water soluble (Ca 2+ + Mg 2+)-activated ATPase has been extracted with 0.1 mM EDTA and 0.1 mM ATP from human erythrocyte membranes. 2. (2) The specific activity of the extracted protein is increased 4- to 6-fold in comparison with untreated ghosts. 3. (3) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [γ- 32P] ATP-labeled erythrocyte membranes shows that the (Ca 2+ + Mg 2+)-activated ATPase is located in the “spectrin” region ( M r 2200 000–240 000). The radioactivity of these high molecular peptide bands is decreased markedly after the extraction of this ATPase at low ionic strength.