A Photochemical Crosslinkinig Study of the Subunit Structure of Membrane‐Associated Spectrin

Abstract

The subunit structure of membrane‐associated spectrin was investigated by crosslinking human erythrocyte membranes with a variety of cleavable photosensitive heterobifunctional reagents by flash photolysis millisecond crosslinking. Crosslinked complexes were analyzed on agarose‐polyacrylamide gels whose high exclusion limit permits the resolution of molecules with M_r larger than 10₆. Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420000, 710000 and 910000, as well as a band at the top of the gels. No significant bands were detectable between the 910000‐M_r band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tetramer, suggesting a limiting tetrameric stoichiometry for membrane‐associated spectrin. The rate of formation of spectrin oligomers was examined by increasing crosslinking time. Dimers accumulate before trimer and tetramer, and as tetramer increased, dimer diminished. A simple kinetic model is employed which is also consistent with spectrin tetramer existing on the cytoplasmic surface of the erythrocyte membrane as the basic structural unit.