Freed B M, Mozayeni B, Lawrence D A, Wallach F R, Lempert N
Cell Immunol. 1986 Aug;101(1):181-94. doi: 10.1016/0008-8749(86)90196-6.
Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.
已知细胞硫醇参与淋巴细胞的激活、分化和生长。理论上，选择性细胞硫醇的烷基化可通过使特定酶或结构蛋白失活来调节激活序列中的特定过程，尽管迄今为止尚未报道过特异性烷基化探针。N-乙基马来酰亚胺（NEM）是一种亲脂性巯基烷基化剂，已知可阻断T淋巴细胞的体外增殖反应。仅在有丝分裂原或同种异体细胞之前或同时添加时，发现NEM（10 microM）在PHA、Con A和MLC试验中具有完全抑制作用；在用PHA刺激细胞仅15秒后添加NEM，导致抑制活性丧失超过50%。用NEM处理细胞10分钟后添加50 microM 2-ME未能阻断抑制作用。NEM（10-20 microM）对淋巴细胞活力没有不良影响，但完全阻断了对有丝分裂原或同种异体细胞的淋巴细胞凝集。在PHA和MLC实验中，淋巴细胞在48小时后克服了NEM的抑制作用。增殖反应的恢复与PHA试验中凝集的开始有关。在使用NEM各种类似物的实验中，我们注意到非极性N-连接侧基的存在是抑制活性所必需的。用NEM预处理PBMC可使总细胞硫醇减少50%，并使增殖阻断99%，而N-羟基马来酰亚胺可使总细胞硫醇减少38%，但对增殖反应没有影响。与NEM反应但不与NHM反应的另外12%的细胞硫醇解释了NEM对淋巴细胞增殖的抑制作用。这些发现表明选择性细胞硫醇对T细胞激活至关重要。
