PRO-TUMOR NECROSIS FACTOR CLEAVAGE ENZYME IN MACROPHAGE MEMBRANE PARTICULATE

Tumour necrosis factor (TNF) is synthesized initially as a membrane-bo und precursor which is then cleaved to yield soluble, mature protein. The 26,000 MW TNF precursor isolated from the lysate of activated RAW 264.7 (mouse macrophage) cells by immunoprecipitation was used to iden tify pro-TNF cleavage enzyme in the same cells. A significant amount o f mature protein was formed in samples containing Nonidet P-40 (NP-40) -lysed cells, whereas sonicated cells showed negligible activity. Most of the cleavage activity in macrophages was localized in the membrane /particulate fraction and remained largely insoluble after sonication or treatment with 2 mm EDTA/1 m NaCl, indicating that the enzyme is as sociated with the membrane/particulate fraction. The crude cleavage ac tivity in membrane/particulate was partially inhibited by a spectrum o f serine, cysteine and aspartate proteinase inhibitors, whereas secret ion of TNF from activated macrophages was inhibited exclusively by ser ine and serine/cysteine proteinase inhibitors. This result suggested t hat, among heterogenous pro-TNF cleavage activities, the enzyme respon sible for the processing of TNF is a serine proteinase. Pro-TNF cleava ge activity was present in non-stimulated macrophages and decreased si gnificantly 8 hr after lipopolysaccharide (LPS) stimulation, suggestin g that it is negatively regulated after an initial burst of TNF synthe sis.