Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin.

J.M Tyler,B.N Reinhardt,D Branton

doi:10.1016/s0021-9258(18)43680-0

Abstract

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin. Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.

Highlights

4.1 have been examined by measuring the binding of and Band 2.1, and between spectrin and Band 4.1. purified ”‘I-Band 2.1 and “%Band 4.1 to [34P]spectrin in solution
Protein A on inactibinding to spectrin arecurvilinear and consistent with vated, lyophilized Staphylococcus aureus cells was from Enzyme a positively cooperative interaction
Together with actin and Band 4.1, spectrin lines the cytoplasmic surface of the red cell membrane with a meshwork that remains afterthe membrane is extracted with nonionic detergents such as TritonX-100 [5,6].Complexes of ing only Bands 1and 2 (Fig. 1).Tetrameric spectrin was obtained by extended dialysis of ghosts at 0-4°C against TES (1 r d a t pH 8.0, EDTA (0.1 m)followed by ultracentrifugation and column chromatography of the supernatant on Sepharose 4B

Summary

Associations of Erythrocyte Membrane Proteins

From the Cell a n d Developmental Biology Department, The Biological Laboratories,Harvard University, Cambridge, Massachusetts 02138. The material was dialyzed against phosphate-buffered saline prior to passing it through either 2.1- or 4.1-Sepharose affinity columns to remove components directedagainstthese polypeptides.Anti-spectrin so purified was treated with iPrrFP' (0.4 mM) and stored a t 0-4'C in a buffer (5 mM NaPO4 at pH 7.6, 1 mM EIITA, 130 mM KCI, 20 mM NaCI, 0.2 mM dithiothreitol, 2 mM NaN:,) suitable for binding assays. After a 90-min incubation, affinity-purified anti-spectrin (40 to 70 pg) was added and the incubation continued for 20 min on ice. Staph A (10% suspension in phosphate-buffered saline with 0.2% Triton X-100 and 1 mg/ml of bovine serum albumin) was added, and the mixturewas centrifuged (8000 rpm, 10 s a t speed) after an additional incubation of 5 to 7 min. Calibration analyses of standard amino acid mixtures were run with the samples ona single ninhydrin batch, and aminoacid concentrations were determined by a Beckman dual channel computing integrator

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DISCUSSION