Yamagata H
J Biochem. 1983 Jun;93(6):1509-15. doi: 10.1093/oxfordjournals.jbchem.a134288.
Escherichia coli mutant Y815 accumulates the precursor of lipoprotein (prolipoprotein) in its envelope. The accumulated prolipoprotein could be chased to mature lipoprotein at 30 degrees C but not at 60 degrees C (Yamagata, H., Ippolito, C., Inukai, M., & Inouye, M. (1982) J. Bacteriol. 152, 1163). When the envelope fraction prepared from the mutant was mixed with the envelope fraction prepared from wild-type E. coli cells and incubated at 60 degrees C in the presence of Triton X-100, the prolipoprotein in the mutant envelope fraction was cleaved rapidly to mature lipoprotein. The cleavage was dependent on the addition of wild-type envelope fraction and Triton X-100 to the reaction mixture. This indicated that the prolipoprotein accumulated in the mutant envelope is a good substrate for the signal peptidase which cleaves the signal peptide from the prolipoprotein, and hence the accumulation of prolipoprotein was due to lack of the signal peptidase in the mutant. The optimum concentration of Triton X-100 for the cleavage of the prolipoprotein in the above in vitro system was 0.05 to 0.1% (v/v) at a wild-type envelope concentration of 0.35 mg protein/ml. Prolipoprotein accumulated in wild-type cells on treatment with globomycin, a specific inhibitor of the signal peptidase, was also cleaved to mature lipoprotein under the same conditions. Triton X-100 was shown to solubilize the signal peptidase from the envelope fraction. The cleavage of the prolipoprotein was rapid and complete in the in vitro system described here, which provides an efficient and convenient assay system for the solubilized signal peptidase for prolipoprotein.
大肠杆菌突变体Y815在其细胞膜中积累脂蛋白前体（前脂蛋白）。积累的前脂蛋白在30℃时可转化为成熟脂蛋白，但在60℃时则不能（山形浩、伊波利托、犬饲正、井上猛（1982年）《细菌学杂志》152卷，1163页）。当将从该突变体制备的细胞膜组分与从野生型大肠杆菌细胞制备的细胞膜组分混合，并在Triton X - 100存在下于60℃孵育时，突变体细胞膜组分中的前脂蛋白迅速裂解为成熟脂蛋白。这种裂解依赖于向反应混合物中添加野生型细胞膜组分和Triton X - 100。这表明在突变体细胞膜中积累的前脂蛋白是信号肽酶的良好底物，该信号肽酶可从前脂蛋白上切割信号肽，因此前脂蛋白的积累是由于突变体中缺乏信号肽酶所致。在上述体外系统中，对于前脂蛋白裂解而言，Triton X - 100的最佳浓度在野生型细胞膜浓度为0.35 mg蛋白质/毫升时为0.05%至0.1%（体积/体积）。用信号肽酶的特异性抑制剂球霉素处理野生型细胞时积累的前脂蛋白，在相同条件下也裂解为成熟脂蛋白。已证明Triton X - 100可从细胞膜组分中溶解信号肽酶。在此所述的体外系统中，前脂蛋白的裂解迅速且完全，这为溶解的前脂蛋白信号肽酶提供了一种高效且便捷的检测系统。
