Lantz M, Lindvall L, Pantazis P, Olsson I
Department of Medicine, University of Lund, Sweden.
Mol Immunol. 1991 Jan-Feb;28(1-2):9-16. doi: 10.1016/0161-5890(91)90081-t.
Production and release of lymphotoxin (LT) was studied by metabolic labeling of human B- and T-cell lines with 14C-leucine and 35S-methionine. LT was immunoprecipitated with antiserum to LT and separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) followed by fluorography. Two molecular weight forms of LT with different rates of release were found both in cell supernatants and cell extracts. Monensin, a sodium ionophore, inhibited the release of LT. LT still appeared in two molecular weight forms after deglycosylation with N-glycanase. Treatment of cells with swainsonine followed by digestion of released LT with endoglycosidase H (endo H) demonstrated that the oligosaccharides were of the complex type. Subcellular fractionation of cells on Percoll density gradients demonstrated that intracellular LT is located to intermediate density fractions. No LT was found in the high density fractions corresponding to lysosomes. Phorbol 12-myristate 13-acetate induced production of tumor necrosis factor (TNF) in the B-lymphoblastoid cell line RPMI-1788. In conclusion, we have demonstrated the presence of two distinct molecular weight forms of LT, which contain N-linked oligosaccharides of the complex type.
通过用¹⁴C-亮氨酸和³⁵S-甲硫氨酸对人B细胞系和T细胞系进行代谢标记，研究了淋巴毒素（LT）的产生和释放。用抗LT抗血清对LT进行免疫沉淀，然后通过十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳（PAGE）进行分离，随后进行放射自显影。在细胞上清液和细胞提取物中均发现了两种释放速率不同的分子量形式的LT。莫能菌素，一种钠离子载体，抑制了LT的释放。用N-聚糖酶去糖基化后，LT仍以两种分子量形式出现。用苦马豆素处理细胞，然后用内切糖苷酶H（endo H）消化释放的LT，结果表明寡糖为复合型。在Percoll密度梯度上对细胞进行亚细胞分级分离表明，细胞内LT定位于中等密度级分。在对应于溶酶体的高密度级分中未发现LT。佛波醇12-肉豆蔻酸酯13-乙酸酯在B淋巴母细胞系RPMI-1788中诱导肿瘤坏死因子（TNF）的产生。总之，我们已经证明存在两种不同分子量形式的LT，它们含有复合型的N-连接寡糖。
