The University of Sydney, Sydney NSW 2006, Australia
Brain spectrin is a major component of the membrane-associated cortical cytoskeleton of neurons and glial cells. In common with other spectrins, it is a highly elongated (~200 nm) protein, comprised of [[alpha]] and [[beta]] subunits that associate laterally in an antiparallel manner to form heterodimers, and 'head-to-head' to form tetramers. The self-association of spectrin dimers to tetramers is crucial to their role as proteins which crosslink actin and membrane-domains; for example, the spectrin dimer is monovalent for actin and therefore incapable of crosslinking actin filaments. While the self-association of the highly specialised erythroid form of spectrin has been studied in detail, little is known about the self-association of the almost ubiquitously expressed non-erythroid forms. In the present study, analytical ultracentrifugation has been used to investigate the self-association of brain spectrin as a function of ionic strength and temperature.
Early studies of brain spectrin using sucrose gradients, electron micrographs and non-denaturing PAGE indicated that the protein was strictly tetrameric, unlike erythroid spectrin which exists in multiple oligomeric forms. Our investigation of the protein using sedimentation equilibrium confirms that brain spectrin is tetrameric in <250 mM NaCl, at 2-25[[ring]]C - the conditions under which the early studies were performed. However, at temperatures above 27[[ring]]C in 100 mM NaCl, sedimentation equilibrium experiments show that brain spectrin forms dimers, tetramers and higher oligomers, as does erythroid spectrin under the same conditions.
Cole and Ralston (1992) found that erythroid spectrin tetramers and higher oligomers were destabilised at 30[[ring]]C with increasing ionic strength above 0.1 M. We have found that brain spectrin is also destabilised by increasing salt, as shown by a transition in the weight average sedimentation coefficient of the protein in 0.2-1.5 M NaCl at 37[[ring]]C. Importantly, similar transitions occur at 20[[ring]]C and 2[[ring]]C, demonstrating that brain spectrin, in marked contrast to erythroid spectrin, undergoes rapid reversible association at temperatures below 25[[ring]]C. An unexpected result was that, while increasing ionic strength at 2-20[[ring]]C destabilises the brain spectrin tetramer, prolonged incubation in 1.5 M NaCl causes indefinite reversible self-association of the protein from the dimer.
Our results show that, in contrast to the indications of previous studies, brain spectrin is not a strictly tetrameric protein, but may behave similarly to erythroid spectrin under physiological conditions.
1. Cole, N & Ralston, G.B. (1992) Biochim. Biophys. Acta 1121, 22-30.