Chinali G
Dipartimento di Medicina Clinica e Sperimentale, Facoltà di Medicina e Chirurgia, Università di Napoli Federico II, Napoles, Italy.
Anal Biochem. 1996 Oct 1;241(1):30-4. doi: 10.1006/abio.1996.0373.
A simple and rapid procedure for the isolation of pure specific complexes of poly(U).ribosome with acylated and/or unacylated tRNA(Phe) is described. The method is based on the use of conditions that favor the formation of polyribosomes. The polyribosomes are separated from ribosomal subunits and monoribosomes by gel filtration on Sepharose 4B at low Mg2+ concentrations that cause the selective dissociation of tRNA-vacant ribosomes from polyribosomes. The procedure allows the isolation in about one hour of various ribosome.poly(U).tRNA complexes completely free of ribosomal particles unable to bind tRNA. A minor fraction of the purified ribosomal complexes is formed by particles devoid of peptidyltransferase activity. Attempts to eliminate this fraction of inactive ribosomes and to obtain ribosomal complexes fully active in polypeptide synthesis were unsuccessful.
本文描述了一种简单快速的方法，用于分离聚（U）·核糖体与酰化和/或未酰化苯丙氨酸tRNA的纯特异性复合物。该方法基于有利于多核糖体形成的条件。在低镁离子浓度下，通过琼脂糖4B凝胶过滤将多核糖体与核糖体亚基和单核糖体分离，低镁离子浓度会导致空载tRNA的核糖体从多核糖体上选择性解离。该方法可在约一小时内分离出各种完全不含无法结合tRNA的核糖体颗粒的核糖体·聚（U）·tRNA复合物。纯化的核糖体复合物中有一小部分是由缺乏肽基转移酶活性的颗粒形成的。消除这部分无活性核糖体并获得在多肽合成中完全有活性的核糖体复合物的尝试未成功。
