Somoskeöy S, Rao M N, Slobin L I
Department of Biochemistry, University of Mississippi School of Medicine, Jackson 39216, USA.
Eur J Biochem. 1996 Apr 1;237(1):171-9. doi: 10.1111/j.1432-1033.1996.0171n.x.
The cytoplasm of mammalian cells of undoubtedly contain a number of different ribonuclease activities, few if any of which have been well characterized. We describe the purification of an exoribonuclease from rabbit reticulocytes which is able to degrade capped RNAs in a 5' to 3' manner. The purified enzyme contains polypeptides of 62 and 58 kDa and may contain an additional polypeptide of 54 kDa. It behaves as a complex of 150 kDa when analyzed by HPLC gel retardation on Superdex 200HR. It is heat-labile, dependent upon divalent cations (Mg2+) for activity, resistant to placental ribonuclease inhibitor, and active over a broad range (10-200 mM) of monovalent cation (K+) concentrations. The enzyme requires a polynucleotide chain of at least 10 bases for activity and cleaves oligonucleotides, up to an octamer long, from the 5' end of an appropriate substrate. In the case of a capped RNA substrate, product analysis by TLC and PAGE indicates that a capped trinucleotide or tetranucleotide or both is produced. Examination of the kinetics of the enzyme with capped and triphosphate-terminated substrates shows that that the cap structure inhibits the action of the enzyme. Furthermore, the data suggest that the rate-limiting step involves the positioning of the enzyme at the 5' end of the substrate and/or cleavage of the first internucleotide bond.
哺乳动物细胞的细胞质无疑含有多种不同的核糖核酸酶活性，但其中几乎没有一种得到很好的表征。我们描述了从兔网织红细胞中纯化一种外切核糖核酸酶的方法，该酶能够以5'至3'的方式降解带帽RNA。纯化后的酶含有62 kDa和58 kDa的多肽，可能还含有一条54 kDa的多肽。当通过Superdex 200HR高效液相色谱凝胶阻滞分析时，它表现为一种150 kDa的复合物。它对热不稳定，活性依赖于二价阳离子（Mg2+），对胎盘核糖核酸酶抑制剂有抗性，并且在较宽范围（10 - 200 mM）的单价阳离子（K+）浓度下都有活性。该酶需要至少10个碱基的多核苷酸链才能发挥活性，并从合适底物的5'端切割寡核苷酸，最长可达八聚体。对于带帽RNA底物，通过薄层层析（TLC）和聚丙烯酰胺凝胶电泳（PAGE）进行产物分析表明会产生一个带帽的三核苷酸或四核苷酸或两者都有。用带帽和三磷酸末端底物研究该酶的动力学表明，帽结构会抑制酶的作用。此外，数据表明限速步骤涉及酶在底物5'端的定位和/或第一个核苷酸间键的切割。
