Krause D, Panet A, Arad G, Dieffenbach C W, Silverman R H
J Biol Chem. 1985 Aug 5;260(16):9501-7.
The regulation of ppp(A2'p)nA-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cells using 2-5A-binding and nuclease activity assays. Minimal levels of 2-5A-dependent RNase were detected in actively dividing clone 1 cells; these levels were independently induced by growth arrest or interferon treatment. Accordingly, levels of the RNase were enhanced during growth arrest by confluency regardless of the presence or absence of interferon or antibody to interferon in the media. Measurement of 2-5A-dependent RNase was unaffected by the addition of any of six different proteinase inhibitors to the cells prior to extraction. The expression of 2-5A-dependent RNase in growth-arrested, interferon-treated cells was still relatively low (about one-third to one-half of that found in similarly treated murine Ehrlich ascites tumor cells). Although this amount of 2-5A-dependent RNase could not be detected by 2-5A-mediated ribosomal RNA cleavage, the activity was identified using a more sensitive novel assay for 2-5A-dependent RNase. In addition, introduction of 2-5A or poly(I) X poly(C) into growth-arrested, interferon-treated cells resulted in some inhibition of protein synthesis. The results indicated that the expression of 2-5A-dependent RNase in NIH 3T3, clone 1 cells is regulated under different physiological conditions and that low levels of 2-5A-dependent RNase were insufficient to significantly inhibit encephalomyocarditis virus replication.
利用2-5A结合和核酸酶活性测定法，在NIH 3T3克隆1细胞中研究了ppp(A2'p)nA-(2-5A)依赖性核糖核酸酶（核糖核酸酶L或核糖核酸酶F）的调节情况。在活跃分裂的克隆1细胞中检测到最低水平的2-5A依赖性核糖核酸酶；这些水平可由生长停滞或干扰素处理独立诱导。因此，无论培养基中是否存在干扰素或抗干扰素抗体，汇合导致的生长停滞期间核糖核酸酶水平都会升高。在提取细胞前添加六种不同的蛋白酶抑制剂中的任何一种，对2-5A依赖性核糖核酸酶的测量均无影响。在生长停滞、经干扰素处理的细胞中，2-5A依赖性核糖核酸酶的表达仍然相对较低（约为经类似处理的小鼠艾氏腹水瘤细胞中发现水平的三分之一至二分之一）。尽管通过2-5A介导的核糖体RNA切割无法检测到这种量的2-5A依赖性核糖核酸酶，但使用一种更灵敏的新型2-5A依赖性核糖核酸酶测定法鉴定出了该活性。此外，将二腺苷5'-磷酸（2-5A）或聚肌苷酸×聚胞苷酸（poly(I)×poly(C)）引入生长停滞、经干扰素处理的细胞中，会导致蛋白质合成受到一定抑制。结果表明，NIH 3T3克隆1细胞中2-5A依赖性核糖核酸酶的表达在不同生理条件下受到调节，且低水平的2-5A依赖性核糖核酸酶不足以显著抑制脑心肌炎病毒复制。
