Herrera J E, Savkur R, Olson M O
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA.
Nucleic Acids Res. 1995 Oct 11;23(19):3974-9. doi: 10.1093/nar/23.19.3974.
Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.
蛋白质B23是一种丰富的核仁蛋白，也是一种假定的核糖体组装因子。使用RNA包埋凝胶和高氯酸沉淀分析法对该蛋白质的核糖核酸酶活性进行了分析。发现三种纯化的细菌表达形式的蛋白质，即B23.1、B23.2和N端带有多组氨酸标签的B23.1以及天然蛋白质都具有核糖核酸酶活性。然而，重组B23.1的比活性大约是重组B23.2的5倍。该活性对人胎盘核糖核酸酶抑制剂不敏感，但被小牛胸腺DNA以剂量依赖的方式抑制。该酶在很宽的pH范围内都有活性，在pH 7.5时表现出明显的最佳活性。低浓度的Ca2+、Mg2+或NaCl的存在能刺激该活性，但不是其活性所必需的。Ca2+的作用是饱和的，且本质上只是刺激性的。相比之下，Mg2+和NaCl表现出刺激活性的最佳浓度，且在高于这些最佳浓度时都会抑制核糖核酸酶活性。这些数据表明蛋白质B23具有内在的核糖核酸酶活性。蛋白质B23在含有前核糖体RNA的核仁亚区室中的定位表明，其核糖核酸酶活性在前核糖体RNA的加工过程中发挥作用。
