Gallis B M, Young E T
J Bacteriol. 1975 May;122(2):719-26. doi: 10.1128/jb.122.2.719-726.1975.
An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.
通过对制备克雷布斯腹水系统的程序进行修改，构建了一种来自酿酒酵母的体外蛋白质合成系统。该蛋白质合成活性由内源性信使指导。氨基酸掺入在较宽的镁和钾浓度范围内发生，分别在6 mM和85 mM时达到最大值。这个体外系统的活性归因于体内起始合成的多肽的延伸。细胞提取物不能利用内源性信使核糖核酸（RNA）起始合成，因为1 μM的放线菌酮（它在体外阻断原核或真核核糖体上的起始）并不能降低氨基酸掺入。然而，10 μM的环己酰亚胺可抑制掺入87%。此外，该系统不受兔网织红细胞多聚核糖体RNA的刺激，兔网织红细胞多聚核糖体RNA在克雷布斯腹水细胞提取物中指导血红蛋白的合成。这种信使的翻译不会被高内源性掺入所掩盖，因为含有[35 - S]甲硫氨酸标记产物的十二烷基硫酸钠 - 聚丙烯酰胺凝胶的放射自显影显示没有合成血红蛋白。该系统的预孵育可将高内源性掺入降低80%，但不会增加其被兔网织红细胞RNA或含酵母聚腺苷酸的RNA刺激的能力。然而，聚尿苷酸确实能刺激聚苯丙氨酸掺入。酵母裂解物不能被添加的天然信使RNA刺激或翻译，对低水平放线菌酮不敏感但被环己酰亚胺抑制，以及其相对较高的镁最佳浓度（与聚尿苷酸相同）表明它能延伸但不能起始多肽链。
