Silverman R H, Jung D D, Nolan-Sorden N L, Dieffenbach C W, Kedar V P, SenGupta D N
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
J Biol Chem. 1988 May 25;263(15):7336-41.
2-5A-dependent RNase (RNase L, RNase F) is an enzyme which mediates effects of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) in cells. 2-5A binding activity present in mouse liver extracts was measured using a 32P-labeled 2-5A derivative. Analysis of Scatchard plots was consistent with a single noninteracting 2-5A binding site with a Ka of 2.5 X 10(10) M-1. Similarly, affinity labeling of proteins with a 32P-labeled 2-5A derivative revealed a single, high-affinity 2-5A-binding protein of Mr 80,000. This 2-5A-binding protein was the only mouse liver protein specifically and consistently eluted by 2-5A from an affinity resin consisting of core(2-5A) covalently attached to cellulose. The 2-5A-eluted protein could degrade polyuridylic acid but not polycytidylic acid. Furthermore, when the 2-5A-eluted protein was electrophoresed into a polyuridylic acid-containing, nondenaturing gel, a band of degraded polyuridylic acid was demonstrated after incubation with 2-5A. There was no band of degraded polyuridylic acid when the elution was performed either in the absence of oligonucleotide or in the presence of low amounts of a closely related analog of 2-5A, p3I2'pA2'pA. Therefore, the Mr 80,000 2-5A-binding protein and the 2-5A-dependent RNase were almost certainly the same protein. Finally, the Mr 80,000 2-5A-binding protein was purified to homogeneity by electroelution from a polyacrylamide gel.
2-5A 依赖性核糖核酸酶（核糖核酸酶 L、核糖核酸酶 F）是一种在细胞中介导 2-5A（px(A2'p)nA；x = 2 或 3，n≥2）作用的酶。使用 32P 标记的 2-5A 衍生物测量小鼠肝脏提取物中存在的 2-5A 结合活性。Scatchard 图分析与一个 Ka 为 2.5×10(10) M-1 的单一非相互作用 2-5A 结合位点一致。同样，用 32P 标记的 2-5A 衍生物对蛋白质进行亲和标记，揭示了一种分子量为 80,000 的单一高亲和力 2-5A 结合蛋白。这种 2-5A 结合蛋白是唯一一种能被 2-5A 从由共价连接到纤维素上的核心(2-5A)组成的亲和树脂中特异性且持续洗脱的小鼠肝脏蛋白。2-5A 洗脱的蛋白可降解聚尿苷酸，但不能降解聚胞苷酸。此外，当将 2-5A 洗脱的蛋白电泳到含聚尿苷酸的非变性凝胶中时，与 2-5A 孵育后显示出一条降解的聚尿苷酸带。当在不存在寡核苷酸或存在少量密切相关的 2-5A 类似物 p3I2'pA2'pA 的情况下进行洗脱时，没有降解的聚尿苷酸带。因此，分子量为 80,000 的 2-5A 结合蛋白和 2-5A 依赖性核糖核酸酶几乎肯定是同一种蛋白。最后，通过从聚丙烯酰胺凝胶中进行电洗脱将分子量为 80,000 的 2-5A 结合蛋白纯化至同质。
