Potenza Nicoletta, Salvatore Vincenzo, Migliozzi Annalucia, Martone Valentina, Nobile Valentina, Russo Aniello
Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy.
Nucleic Acids Res. 2006 May 31;34(10):2906-13. doi: 10.1093/nar/gkl368. Print 2006.
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/K(m) 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.
人核糖核酸酶-1（hRNase-1）是一种细胞外酶，存在于外分泌胰腺、血液、乳汁、唾液、尿液和精液中，它与膳食RNA的消化以及抗病毒宿主防御有关。该酶的特点是对单链和双链RNA都具有高催化活性。在本研究中，我们探讨了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杂交体中的RNA成分。为此，我们基于一种由12个核苷酸的5'-荧光素标记的RNA与互补的3'-淬灭剂修饰的DNA杂交而成的荧光底物，开发了一种准确且灵敏的实时核糖核酸酶H检测方法。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，其催化常数与米氏常数之比（kcat/K(m)）为330000 M⁻¹ s⁻¹，该值比同源的牛核糖核酸酶A高出180多倍，仅比大肠杆菌核糖核酸酶H测得的值低8倍。hRNase-1的动力学特征表明，其杂交酶活性在中性pH时最大，随离子强度降低而增加，并被胞质核糖核酸酶抑制剂完全抑制。总体而言，所报道的数据拓宽了我们对hRNase-1酶学性质的认识，并为理解其生物学功能提供了新的线索。
