sepharose and Rheumatic-Diseases

A rapid method has been developed for enrichment of Jo-1 antigen (histidyl-tRNA synthetase) from HeLa cells. The enzyme has been prepared from post-ribosomal supernatant by successive chromatography with Blue Sepharose and Poly-U-Sepharose, followed by DEAE-high performance liquid chromatography (HPLC). By this method, enzyme could be obtained within 4 days of HeLa cell harvesting, with 40% recovery of the enzymatic activity. The apparent native molecular size of the enzyme as determined by HPLC-size exclusion column chromatography was approximately 120 kDa. Under denaturing conditions using SDS-polyacrylamide gel electrophoresis the enzyme subunit size was approximately 55 kDa. The antigen preparation, although not homogeneous, was found to react only with anti-Jo-1 positive antisera when tested by immunoblotting with many patient sera of defined autoantibody specificities, making the preparation useful for immunologic studies of anti-Jo-1 antibodies.

Topics: Amino Acyl-tRNA Synthetases; Antibodies, Antinuclear; Autoantigens; Autoimmune Diseases; Chromatography, Gel; Chromatography, High Pressure Liquid; Collodion; Electrophoresis, Polyacrylamide Gel; HeLa Cells; Histidine-tRNA Ligase; Humans; Molecular Weight; Rheumatic Diseases; Sepharose