RESUMO
PURPOSE: A variety of methods have been developed for the purification of S-antigen but a simple and rapid procedure based on hydroxyapatite-agarose (HA) adsorbtion is most widely used. In the present study, we investigated the nature of proteins purified with the aid of HA chromatography. METHODS: After elimination of retinal S-antigen by HA, the soluble extract of retinal tissue was readsorbed on HA. The proteins were thereafter desorbed by 10 to 500 mM phosphate buffer gradient. Two peaks obtained by SDS-PAGE were used for the generation of specific antisera for subsequent analysis by ELISA and Western blotting. RESULTS: Four proteins (two 48 kDa , one 50 kDa and one 46 kDa) were obtained in this manner. Partial amino acid sequencing permitted the identification of these proteins as alpha-enolase (48 kDa), gamma-enolase (48 kDa), Glucose-6-phosphate-Isomerase (50 kDa) and aspartate-amino-transferase (46 kDa). CONCLUSION: The selected glycolytic enzymes co-purified with retinal S-antigen by hydroxyapatite agarose chromatography of bovine retina.
Assuntos
Arrestina/isolamento & purificação , Cromatografia em Agarose/métodos , Durapatita , Enzimas/isolamento & purificação , Glicólise , Retina/química , Sequência de Aminoácidos/genética , Animais , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/isolamento & purificação , Bovinos , Enzimas/genética , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/isolamento & purificação , Glicólise/fisiologia , Dados de Sequência Molecular , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificaçãoRESUMO
A procedure is described for the rapid isolation of S antigen from retinal tissue, by salt precipitation, gel filtration and final purification by adsorption chromatography on hydroxyapatite-agarose. The method yielded a highly purified material and allowed an excellent preservation of the antigenicity, as shown by high pathogenic activity in induction of experimental autoimmune uveoretinitis.