Formation of 100 Å filaments from purified glial fibrillary acidic protein in Vitro

Abstract

Glial fibrillary acidic protein, which is specific to astroglia in the central nervous system, polymerizes in vitro into filaments similar to native ~ 100 Å filaments. Following purification from aqueous extracts of bovine brain by immunoaffinity chromatography, GFA † † Abbreviations used: GFA, glial fibrillary acidic; SDS, sodium dodecyl sulfate. protein is highly soluble in very low ionic strength solutions. Sedimentation equilibrium analysis of protein solutions in prefilament solvent conditions (2 m m-Tris · HCl, pH 7.8, 20 °C, containing 0.5 m m-dithiothreitol) indicates a paucidisperse mixture of species in solution with a typical range of apparent weight-average molecular weights from about 186,000 to 227,000. Between pH 6.0 and 8.0 the solubility is a function of pH and ionic strength as well as temperature, and precipitation is favored by lowering the pH or temperature and by raising the ionic strength. GFA protein associates in the form of filaments over a narrow range of pH and ionic strength; optimal conditions for polymerization of a 0.1 mg/ml protein solution are 100 m m-imidazole-HCl buffer (pH 6.8), at a temperature of 37 °C, and there is no requirement for co-factors. Filaments appear primarily as tangles of smooth curvilinear structures approximately 100 Å in diameter and of indefinite length, although some lateral association of filaments into thick bundles is also observed. While the formation of filaments is not affected by the presence or absence of reducing agent, under oxidizing conditions disulfide linkages form between protein subunits. Disassembly is achieved by dialysis against 2 m m-Tris · HCl buffer (pH 8.5), but this process is significantly enhanced by the addition of 0.5 mM-dithiothreitol during assembly and disassembly. These experiments clarify the role of GFA protein as the subunit of astroglialspecific intermediate filaments. In addition, they suggest that the 100 Å filament, as other components of the cytoskeleton, may assemble and disassemble in the glial cytoplasm.