SEPARATION OF TUMOR-NECROSIS-FACTOR-ALPHA ISOFORMS BY 2-DIMENSIONAL POLYACRYLAMIDE-GEL-ELECTROPHORESIS

The mouse macrophage cell-line RAW264.7, stimulated with lipopolysacch aride, was used as a model for the study of the production of tumor ne crosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a p rotease to release a mature molecule of 17 kDa. Dose-dependent product ion of transmembrane TNF was assessed by fractionation of cell membran es and Western blot analysis followed by autoradiography and densitome try. Isoforms of both the precursor and mature molecules were separate d using two-dimensional (2-D) electrophoresis with immobilised pH grad ient 3-10 linear gels as the first dimension. After radiolabelling of cells with S-35, both cell-associated and supernate-associated TNF iso forms were immunoprecipitated. A large number of protein spots were vi sualised on the 2-D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one-dimensiona l sodium dodecyl sulphale-polyacrylamide gel electrophoresis (SDS-PAGE ). The likelihood that these putative isoforms were the result of diff erential glycosylation was tested by preincubating the cells with tuni camycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of pr ecursor TNF isoforms that were unchanged upon tunicamycin treatment an d these presumably reflect protein modifications other than glycosylat ion.