A photochemical crosslinking study of the subunit structure of membrane-associated spectrin.

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 the resolution of molecular with Mr larger than 10(6). Crosslinking of membrane ghosts produced a series of new bands with apparent molecular weights of approximately 420 000, 170 000 and 910 000, as well as a band at the top of the gels. No significant bands were detectable between the 910 000-Mr band and the material at the gel top. The molecular weights of the three bands correspond to spectrin dimer, trimer, and tatramer, 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.