Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay.
Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay.
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DOI：
10.1093/nar/gkl368
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发表时间：
2006
影响因子：
14.9
通讯作者：
Russo A
中科院分区：
文献类型：
--
作者：
Potenza N;Salvatore V;Migliozzi A;Martone V;Nobile V;Russo A
人核糖核酸酶-1（human ribonuclease-1，hRNase-1）是一种存在于胰腺、血液、乳汁、唾液、尿液和精浆等外分泌系统中的胞外酶，参与食物RNA的消化和抗病毒宿主防御。该酶的特征在于对单链和双链RNA都具有高催化活性。在这项研究中，我们探索了hRNase-1也可能具有核糖核酸酶H活性的可能性，即能够消化RNA：DNA杂合体的RNA组分。为此，我们开发了一种准确、灵敏的实时RNase H检测方法，该方法基于由12 nt 5′-荧光素标记的RNA与互补的3′-淬灭剂修饰的DNA杂交制成的荧光底物。在类似生理条件下，发现hRNase-1能非常有效地切割RNA：DNA杂交体，其kcat/Km值为330 000 M−1 s−1，比用同源牛RNase A测得的值高180倍以上，仅比用大肠杆菌RNase H测得的值低8倍。hRNase-1的动力学特性表明，其杂交酶活性在中性pH下最大，随着离子强度的降低而增加，并且被胞质RNase抑制剂完全抑制。总之，报道的数据拓宽了我们对hRNase-1的酶性质的了解，并为理解其生物学功能提供了新的元素。
Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5′-fluorescein-labeled RNA hybridized to a complementary 3′-quencher-modified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a kcat/Km of 330 000 M−1 s−1, a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.
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