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[来自玉米幼苗的无细胞蛋白质合成系统]

[A cell-free system of protein synthesis from maize seedlings].

作者信息

Sim W S, Klämbt D

机构信息

Botanisches Institut der Universität Bonn, Meckenheimer Allee 170, D-5300, Bonn, Federal Republic of Germany.

出版信息

Planta. 1973 Jun;113(2):157-71. doi: 10.1007/BF00388200.

Abstract

An amino acid incorporating system has been prepared from maize seedlings, and it has been characterized with the aid of poly-U and a "mRNA enriched fraction" from the same plant material.The rate of protein synthesis decreases proportionally with the incubation period. It seems to be related to the degradation of polysomes. The optimal Mg(2+) concentration is 20 mM for the poly-U dependent protein synthesis and 10 mM for the synthesis with endogenous polysomes. The poly-U directed polyphenylalanine synthesis is increased 12-fold by addition of exogenous sRNA. Under optimal conditions poly-U causes a 40-fold increase of the phenylalanine incorporation.A "mRNA enriched fraction" was prepared from maize seedlings using proteinase K for deproteination of polysomes. The resulting RNA was further fractionated by successive precipitation with LiCl, NaCl and ethanol and characterized by polyacrylamide gel electrophoresis. The addition of 57 μg of the mRNA-enriched sample increases the incorporation of amino acid into polypeptides by a factor of approximately 2 at a Mg(2+) concentration of 5 mM, and by a factor of 1.5 at 15 mM Mg(2+). The addition of 72 μg rRNA does not stimulate the incorporation at low Mg(2+) concentration, while at 15 mM Mg(2+) a 1.3-fold increase is observed.

摘要

已从玉米幼苗中制备了一个氨基酸掺入系统,并借助多聚尿苷(poly-U)和来自同一植物材料的“富含mRNA的组分”对其进行了表征。蛋白质合成速率与孵育时间成比例下降。这似乎与多核糖体的降解有关。对于依赖多聚尿苷的蛋白质合成,最佳镁离子(Mg(2+))浓度为20 mM,而对于利用内源性多核糖体的合成,最佳镁离子浓度为10 mM。添加外源性可溶性RNA(sRNA)可使多聚尿苷指导的聚苯丙氨酸合成增加12倍。在最佳条件下,多聚尿苷可使苯丙氨酸掺入量增加40倍。使用蛋白酶K对玉米幼苗的多核糖体进行脱蛋白处理,制备了一个“富含mRNA的组分”。所得RNA通过依次用氯化锂(LiCl)、氯化钠(NaCl)和乙醇沉淀进一步分级分离,并通过聚丙烯酰胺凝胶电泳进行表征。在镁离子浓度为5 mM时,添加57 μg富含mRNA的样品可使氨基酸掺入多肽的量增加约2倍,在镁离子浓度为15 mM时增加1.5倍。添加72 μg核糖体RNA(rRNA)在低镁离子浓度下不会刺激掺入,而在镁离子浓度为15 mM时,掺入量增加1.3倍。

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