Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction.
Exposure of human erythrocyte ghosts (pH 8, 10 degrees C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent. Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with N-ethylmaleimide, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH2 groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141--151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH2 groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH2 groups are not involved in the reaction.
D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000528:radiation effects; D004912:Erythrocytes / Q000378:metabolism*; D005021:Ethylamines / Q000494:pharmacology; D006801:Humans; D008027:Light; D008565:Membrane Proteins / Q000378:metabolism*; D008751:Methylene Blue / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010777:Photochemistry; D013049:Spectrin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D005021:Ethylamines; D008565:Membrane Proteins; D013049:Spectrin; D008751:Methylene Blue