TUMOR NECROSIS FACTOR-DRIVEN FORMATION OF DISULFIDE-LINKED RECEPTOR AGGREGATES

We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been pa rtially purified by gel filtration chromatography yielded two prominen t bands of M(r) 60,000 and 75,000 corresponding to the two known TNFR (types I and II, respectively). In addition to these, types I and II T NFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting th at TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that h ad been partially purified by gel filtration chromatography and incuba ted with TNF before electrophoretic separation. Indeed, type II TNFR a ggregates, but not type I TNFR aggregates, were generated at optimal T NF concentrations. Formation of type II TNFR aggregates in this last e xperimental setting and of both type I and type II TNFR aggregates dur ing affinity purification could be prevented if an alkylating agent (N -ethylmaleimide) was added during the TNFR-TNF incubation step. Simila r results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggr egates were generated at TNF concentrations ranging from 10(-9) to 10( -10) M and their formation was prevented in the presence of N-ethylmal eimide.