盘基网柄菌核糖核酸酶P的部分纯化及特性分析

Partial purification and characterization of RNase P from Dictyostelium discoideum.

作者信息

Stathopoulos C, Kalpaxis D L, Drainas D

机构信息

Laboratory of Biological Chemistry, School of Medicine, University of Patras, Rio-Patras, Greece.

出版信息

Eur J Biochem. 1995 Mar 15;228(3):976-80. doi: 10.1111/j.1432-1033.1995.tb20349.x.

DOI:10.1111/j.1432-1033.1995.tb20349.x

PMID:7737203

Abstract

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S. pombe RNase P, producing the mature 5' end of tRNA(Ser). pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl. The apparent Km for the S. pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.

摘要

来自盘基网柄菌的核糖核酸酶P（RNase P）已被纯化了470倍。盘基网柄菌RNase P在与粟酒裂殖酵母RNase P相同的位点切割粟酒裂殖酵母抑制性tRNA（Ser）的前体，产生tRNA（Ser）的成熟5'末端。酶活性的最适pH和温度分别为7.6和37℃。该酶在5 mM MgCl2和10 mM NH4Cl或5 mM KCl存在下表现出最佳活性。源自supS1 tRNA（Ser）基因的粟酒裂殖酵母tRNA前体的表观Km为240 nM，表观Vmax为3.6 pmol/min。蛋白酶K和微球菌核酸酶对盘基网柄菌RNase P的抑制强烈表明该活性需要蛋白质和RNA组分。在硫酸铯密度梯度中，该酶的浮力密度为1.23 g/ml，表明全酶的RNA/蛋白质比率较低。