Glial fibrillary acidic protein from normal human brain. Purification and properties

Abstract

The glial fibrillary acidic (GFA) protein was purified from normal human brain and gliosed material (white matter with multiple sclerosis plaques) and itsproperties were compared with those of other brain specific proteins in the literature. SDS-acrylamide gel electrophoresis and immunodiffusion with GFA protein antiserum were used to monitor each step of the purification procedure. Both water-soluble and detergent-soluble fractions of GFA protein reacted with antibodies to the soluble fraction giving an immunodiffusion pattern of complete identity. Purified preparations of the water-soluble fraction were either obtained with differential salt extraction followed by DEAE-Sephadex and phosphocellulose column chromatography, or with 25% ammonium sulfate precipitation and pH 5.0 acid precipitation followed by DEAE-Sephadex column chromatography. Depending on the material used as a source ( i.e., normal or gliosed material) preparationns of 85–92% purity were obtained, as indicated by the relative dye binding on SDS-acrylamide gels. The particulatebound fraction was purified from gliosed material by 30% ammonium sulfate precipitation followed by 2 cycles of Sephadex G-200 gel filtration. Immunization of rabbits with aqueous extracts of normal human white matter produced antisera which gave one main precipitation line when tested by immunodiffusion against normal brain extracts. When these anti-white matter immune sera and GFA protein antisera were tested against normal brain extracts and purified GFA protein preparations, precipitation lines were obtained with an immunodiffusion pattern of complete identity, indicating that GFA protein was the main antigen identified by reacting anti-white matter immune sera with normal brain extracts.