Bayard B, Zhou A
CNRS U.A. 530, USTL, Montpellier, France.
Anal Biochem. 1992 Jan;200(1):108-14. doi: 10.1016/0003-2697(92)90284-e.
The intracellular effectors known as 2-5A (ppp-(A2'p)nA) regulate the cleavage of single-stranded RNA by activating a latent endoribonuclease (2-5A-dependent RNase or RNase L). Accordingly this enzyme may exist in either an inactive form, free of 2-5A, or an active, 2-5A-bound form. Previously, a radiobinding assay for 2-5A-dependent RNase was developed that measured the amount of labeled ppp(A2'p)2A3'-[32P]Cp, a derivative of ppp(A2'p)nA, that bound the inactive enzyme form. Because 2-5A-dependent RNase has a particularly high affinity for 2-5A the radiobinding assay may not measure the 2-5A activated form of the enzyme. Therefore an efficient procedure to facilitate the detection of total 2-5A-dependent RNase (i.e., 2-5A-free and 2-5A-bound enzyme) in mouse spleen extracts was developed. Denaturing conditions were used to ensure that all 2-5A-dependent RNase was in the 2-5A-free form. After denaturation on polyacrylamide gel electrophoresis, optimal blotting conditions onto nitrocellulose and renaturation of the 2-5A binding site of 2-5A-dependent RNase were developed. This procedure allowed a population of enzyme that otherwise is not accessible by the classical radiobinding assay to be assayed, thus leading to an increased measurement of 15-17% in cytoplasmic spleen extracts.
被称为2-5A（ppp-(A2'p)nA）的细胞内效应物通过激活一种潜在的核糖核酸内切酶（2-5A依赖性核糖核酸酶或核糖核酸酶L）来调节单链RNA的切割。因此，这种酶可能以两种形式存在，一种是无2-5A的无活性形式，另一种是与2-5A结合的活性形式。以前，人们开发了一种针对2-5A依赖性核糖核酸酶的放射结合测定法，该方法可测量与无活性酶形式结合的标记的ppp(A2'p)2A3'-[32P]Cp（ppp(A2'p)nA的衍生物）的量。由于2-5A依赖性核糖核酸酶对2-5A具有特别高的亲和力，放射结合测定法可能无法测量该酶的2-5A激活形式。因此，人们开发了一种有效的程序来促进检测小鼠脾脏提取物中总的2-5A依赖性核糖核酸酶（即无2-5A和与2-5A结合的酶）。使用变性条件以确保所有2-5A依赖性核糖核酸酶都处于无2-5A的形式。在聚丙烯酰胺凝胶电泳上变性后，开发了将其转移至硝酸纤维素膜上的最佳印迹条件以及2-5A依赖性核糖核酸酶的2-5A结合位点的复性条件。该程序使得经典放射结合测定法无法检测到的一部分酶能够被检测，从而使细胞质脾脏提取物中的测量值增加了15%-17%。
