Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates

Laura Grazioli, Daniele Casero, Alessandra Restivo, Elena Cozzi, Fabrizio Marcucci

Research output: Contribution to journalArticle

Abstract

We have characterized, by ligand blotting, solubilized tumor necrosis factor receptors (TNFR) from K562 cells. Preparations that had been partially purified by gel filtration chromatography yielded two prominent 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 TNFR-related species of M(r) > 100,000 were detected after purification by tumor necrosis factor (TNF)-affinity chromatography, suggesting that TNF had driven receptor aggregation during this step. To test this hypothesis ligand blots were performed on receptor preparations that had been partially purified by gel filtration chromatography and incubated with TNF before electrophoretic separation. Indeed, type II TNFR aggregates, but not type I TNFR aggregates, were generated at optimal TNF concentrations. Formation of type II TNFR aggregates in this last experimental setting and of both type I and type II TNFR aggregates during affinity purification could be prevented if an alkylating agent (N-ethylmaleimide) was added during the TNFR-TNF incubation step. Similar results were obtained when intact K562 cells were incubated with TNF and then analyzed for receptor aggregation; type II TNF receptor aggregates were generated at TNF concentrations ranging from 10-9 to 10- 10 M and their formation was prevented in the presence of N-ethylmaleimide.

Original languageEnglish
Pages (from-to)22304-22309
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number35
Publication statusPublished - Sep 2 1994

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Receptors, Tumor Necrosis Factor, Type II
Disulfides
Receptors, Tumor Necrosis Factor, Type I
Tumor Necrosis Factor-alpha
Receptor Aggregation
Ethylmaleimide
K562 Cells
Tumor Necrosis Factor Receptors
Chromatography
Purification
Gel Chromatography
Agglomeration
Gels
Ligands
Affinity chromatography
Alkylating Agents
Affinity Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Grazioli, L., Casero, D., Restivo, A., Cozzi, E., & Marcucci, F. (1994). Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates. Journal of Biological Chemistry, 269(35), 22304-22309.

Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates. / Grazioli, Laura; Casero, Daniele; Restivo, Alessandra; Cozzi, Elena; Marcucci, Fabrizio.

In: Journal of Biological Chemistry, Vol. 269, No. 35, 02.09.1994, p. 22304-22309.

Research output: Contribution to journalArticle

Grazioli, L, Casero, D, Restivo, A, Cozzi, E & Marcucci, F 1994, 'Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates', Journal of Biological Chemistry, vol. 269, no. 35, pp. 22304-22309.
Grazioli L, Casero D, Restivo A, Cozzi E, Marcucci F. Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates. Journal of Biological Chemistry. 1994 Sep 2;269(35):22304-22309.
Grazioli, Laura ; Casero, Daniele ; Restivo, Alessandra ; Cozzi, Elena ; Marcucci, Fabrizio. / Tumor necrosis factor-driven formation of disulfide-linked receptor aggregates. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 35. pp. 22304-22309.
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