Abstract
The complete primary structure of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations has been determined. The sequence of this domain, which contains 67 amino acids and has a molecular mass of 8045 daltons, has been obtained by NH2-terminal sequence analysis of an 8-kDa chymotryptic peptide, three endoproteinase lysine C-cleaved peptides and two peptides obtained by Staphylococcus aureus protease V8 cleavage. All peptides including the 8-kDa domain peptide were purified by reverse-phase high performance liquid chromatography. Antibodies against two different synthetic peptides of the 8-kDa domain are able to inhibit the association between protein 4.1, spectrin, and F-actin, corroborating that the 8-kDa domain is responsible for the formation of a ternary complex. A computer search of the 8-kDa sequence with the National Biomedical Research Foundation database did not detect any significant homologies to known sequences. Protein 4.1 is not related to any known proteins and may represent a new protein superfamily.
Highlights
The red cell membrane has been chosen as a model for studying membrane-cytoskeleton interactions because of its apparent simplicity relative toother cells
The precise nature of the ternary complex is not yet well understood, it is likely to be of general significance since proteins immunologically related to erythrocyte spectrinandprotein 4.1 have been described in numerous non-erythroid cells
This fragment is incorporated into theternary complex in approximately stoichiometric amounts and its activity is comparable to thatof the intact4.1 molecule on a molar basis [14]
Summary
Isohtion of a Complex-promoting Peptide of Protein4.1”Two different methods have been followed for the isolation of the 8-kDa domain of erythrocyteprotein 4.1. Protein 4.1 (2 mg) wasdigested with a-chymotrypsin at 1:260 enzyme to substrate ratio and incubated with spectrin (12 mg) and F-actin (8 mg) for 90 min at 0 “C.After centrifugation at 150,000X g for 30 min on a SW50.1 rotor, the 8-kDa peptide obtained in the pellet fraction was chromatographed on gel filtration columns (two 30-cm Bio-Si1 TSK-400 columns, two 30-cm Bio-Si1TSK-250 columns, and one 30cm Bio-Si1TSK-125 column, all in tandem,Bio-Rad) equilibrated in 8 M urea, 0.2 M Tris, 10mM 2-mercaptoethanol, pH 7.0. Nomenclature of Peptides-Peptides were named based on the type of cleavage used endoprotease lysine-C (EL); S. aureus protease V8 (SV8) Each of these two sets of peptides have been numbered in the order which they occur in the complete sequence starting at the amino-terminal. Peptide A included amino acids 1-15 and peptide B amino acids 46-59 (Fig. 4) Both peptides were independently coupled to KLH (keyhole limpet hemocyanine) using the bifunctional reagent rn-maleimidobenzoylsulfosuccinimide esteirn 50 mM phosphate buffer, 1 mM EDTA, pH 7.0N.
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