Pro-tumour necrosis factor cleavage enzyme in macrophage membrane/particulate.
Pro-tumour necrosis factor cleavage enzyme in macrophage membrane/particulate.
复制标题
巨噬细胞膜/颗粒中的促肿瘤坏死因子裂解酶。
作者：
K. Kim;O. Kwon;D. Jue
肿瘤坏死因子(TNF)最初是作为膜结合的前体合成的，然后被切割成可溶的成熟蛋白。用免疫沉淀法从活化的RAW 264.7(小鼠巨噬细胞)的裂解物中分离出26,000 mW的肿瘤坏死因子前体，用于鉴定同一细胞中的肿瘤坏死因子前体。在含有NONIDT P-40(NP-40)裂解细胞的样本中形成了相当数量的成熟蛋白，而超声处理的细胞显示出很小的活性。巨噬细胞的大部分切割活性定位于膜/颗粒组分，经超声或2 mM EDTA/1M氯化钠处理后仍基本不溶，表明该酶与膜/颗粒组分有关。丝氨酸、半胱氨酸和天冬氨酸蛋白酶抑制剂部分抑制膜/颗粒的粗裂解活性，而激活的巨噬细胞分泌肿瘤坏死因子仅被丝氨酸和丝氨酸/半胱氨酸蛋白酶抑制剂抑制。这一结果表明，在异源的前肿瘤坏死因子的切割活性中，负责处理肿瘤坏死因子的酶是一种丝氨酸蛋白酶。未受刺激的巨噬细胞中存在肿瘤坏死因子原的裂解活性，在内毒素刺激后8小时显著降低，提示在肿瘤坏死因子合成的最初爆发后，它被负调控。
Tumour necrosis factor (TNF) is synthesized initially as a membrane-bound 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 identify pro-TNF cleavage enzyme in the same cells. A significant amount of 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 associated with the membrane/particulate fraction. The crude cleavage activity in membrane/particulate was partially inhibited by a spectrum of serine, cysteine and aspartate proteinase inhibitors, whereas secretion of TNF from activated macrophages was inhibited exclusively by serine and serine/cysteine proteinase inhibitors. This result suggested that, among heterogenous pro-TNF cleavage activities, the enzyme responsible for the processing of TNF is a serine proteinase. Pro-TNF cleavage activity was present in non-stimulated macrophages and decreased significantly 8 hr after lipopolysaccharide (LPS) stimulation, suggesting that it is negatively regulated after an initial burst of TNF synthesis.