Isolation and characterization of peptides derived from the cytoplasmic segment of band 3, the predominant intrinsic membrane protein of the human erythrocyte.

Abstract

An improved method for the large scale isolation of Band 3 has been developed and used to isolate peptides from the cytoplasmic portion of the molecule. Membranes were treated with 33 mM lithium 3,5-diiodosalicylate to remove “extrinsic” membrane proteins and then solubilized in 2% sodium dodecyl sulfate. An activated thiol column was used to separate the sialoglycoproteins from Band 3 and the latter was purified by gel filtration on Sepharose 6B in sodium dodecyl sulfate. A chymotryptic fragment of Band 3 (M, 58,000; chymotryptic Band 3), produced by digestion of intact cells, and a tryptic fragment of Band 3 WI, 49,000; tryptic Band 3), produced by digestion of membranes were purified similarly. Each purified component was cleaved by S-cyanylation (Jacobson, G. R., Schaffer, M. H., Stark, G. R., and Vanaman, T. C. (1973) J. Biol. Chem. 248, 6583-6591) in 7.5 M guanidine hydrochloride. Intact and chymotryptic Band 3 produced three peptides (iM, 36,000, 24,000, and 12,000) which appeared to be homogeneous when analyzed by gel electrophoresis in sodium dodecyl sulfate or acid-urea. Lactoperoxidase-catalyzed iodination indicated that these peptides are derived from the cytoplasmic side of membrane. These peptides were purified by CM-cellulose and Sephadex G-100 and found to be rich in glutamate (or glutamine) and did not contain detectable sugars. Antibodies prepared against Band 3 were found to be directed against the cytoplasmic segment of Band 3 and these were used to show that the 12,000and 24,000-dalton peptides were derived from the 36,000-dalton piece. Cleavage of intact Band 3 by a two-step cyanylation reaction using K’%N indicated that the 36,000dalton peptide and the 24,000-dalton peptide were both derived from the NH,-terminal end. These experiments support the idea that a 40,000-dalton segment of Band 3 bearing the NH, terminus is on the cytoplasmic side of the lipid bilayer. These results are also consistent with the view that the bulk of the Band 3 material is a homogeneous polypeptide chain.

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@article{Fukuda1978IsolationAC, title={Isolation and characterization of peptides derived from the cytoplasmic segment of band 3, the predominant intrinsic membrane protein of the human erythrocyte.}, author={Mizue Fukuda and Yuval Eshdat and Guido Tarone and Vincent T. Marchesi}, journal={The Journal of biological chemistry}, year={1978}, volume={253 7}, pages={2419-28} }