Tokunaga H, Wu H C
J Biol Chem. 1984 May 25;259(10):6098-104.
We have previously shown that an Escherichia coli mutant ( mlpA allele) containing a structurally altered murein prolipoprotein due to substitution of Gly14 by Asp14 , is globomycin resistant. In addition, the mutant prolipoprotein is not modified with glyceride and consequently remains uncleaved. Spontaneous revertants possessing a mature lipoprotein of apparent normal structure can be isolated by EDTA selection. Three revertants were chosen in the present study which included the analysis of kinetics of lipoprotein maturation and the determination of globomycin sensitivity. These pseudorevertants in the lpp gene which could be recognized by the anomalous prolipoprotein mobility in sodium dodecyl sulfate gels, exhibited altered globomycin sensitivity in vivo. Our results indicate that alterations in prolipoprotein structure affect the kinetics of prolipoprotein modification and processing reactions, both in vivo and in vitro. Pulse-chase experiments revealed the transient existence of unmodified prolipoprotein and modified prolipoprotein as biosynthetic intermediates of mature lipoprotein. The rate of prolipoprotein modification appeared to be slightly faster than that of processing in the wild type cell. In contrast, modification of prolipoprotein was rate limiting in a pseudorevertant strain 14R21 , and the processing of 14R21 modified prolipoprotein appeared to proceed more rapidly than that of wild type prolipoprotein, both in vitro and in vivo.
我们先前已表明，一种大肠杆菌突变体（mlpA等位基因），由于其14位甘氨酸被天冬氨酸取代，导致其胞壁质前脂蛋白结构发生改变，对球霉素具有抗性。此外，该突变体前脂蛋白未被甘油酯修饰，因此仍未被切割。通过EDTA筛选可分离出具有明显正常结构的成熟脂蛋白的自发回复突变体。本研究选择了三个回复突变体，包括对脂蛋白成熟动力学的分析以及对球霉素敏感性的测定。这些lpp基因中的假回复突变体在十二烷基硫酸钠凝胶中可通过异常的前脂蛋白迁移率识别，在体内表现出改变的球霉素敏感性。我们的结果表明，前脂蛋白结构的改变会影响体内和体外前脂蛋白修饰及加工反应的动力学。脉冲追踪实验揭示了未修饰的前脂蛋白和修饰的前脂蛋白作为成熟脂蛋白生物合成中间体的短暂存在。在野生型细胞中，前脂蛋白修饰的速率似乎略快于加工速率。相比之下，在假回复突变体菌株14R21中，前脂蛋白的修饰是限速步骤，并且在体外和体内，14R21修饰的前脂蛋白的加工似乎比野生型前脂蛋白进行得更快。
