Sutrina S L, Chin A M, Esch F, Saier M H
Department of Biology, University of California, San Diego, La Jolla 92093.
J Biol Chem. 1988 Apr 15;263(11):5061-9.
The proteins comprising the fructose-specific phosphoenolpyruvate:sugar phosphotransferase system were investigated using a strain of Salmonella typhimurium which lacks the general phosphotransferase system proteins, HPr and Enzyme I, synthesizes the fructose phosphotransferase system proteins, FPr, Enzyme IIfru, Enzyme IIIfru, and fructose-1-phosphate kinase, constitutively, and expresses the Enzyme I-like protein Enzyme I. Enzyme I activity was found in the cytoplasmic fraction, Enzyme IIfru in the membrane fraction, and FPr and Enzyme IIIfru activities were distributed between the two fractions. Extraction of membranes with butanol and urea led to quantitative release of the membrane-associated Enzyme IIIfru and FPr activities, while Enzyme IIfru remained with the membranes. FPr was purified to homogeneity using ion exchange chromatography, gel filtration, and reversed phase high pressure liquid chromatography (HPLC), and its amino acid composition and N-terminal sequence were determined. A complex of FPr and Enzyme IIIfru (Mr 50,000) was also purified to near homogeneity using ion exchange chromatography, gel filtration, and chromatography on hydroxylapatite. When the purified complex was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was visualized as two protein bands with mobilities corresponding to molecular weights of about 40,000 (Enzyme IIIfru) and 9,000 (FPr). Neither the FPr and Enzyme IIIfru activities nor the proteins represented by these two bands separated during the above chromatography steps or using any of several other techniques, including reversed phase HPLC, indicating a very tight association. Active Enzyme IIIfru free of FPr was never isolated or observed. The proteins could be separated in denatured form by gel filtration in the presence of guanidine HCl or urea. Free FPr and the FPr-Enzyme IIIfru complex were characterized, and the properties of free and complexed FPr were compared to those of HPr.
糖磷酸转移酶系统的蛋白质进行了研究。该菌株缺乏通用磷酸转移酶系统蛋白HPr和酶I，组成型合成果糖磷酸转移酶系统蛋白FPr、酶IIfru、酶IIIfru和果糖-1-磷酸激酶，并表达类酶I蛋白酶I。酶I活性存在于细胞质部分，酶IIfru存在于膜部分，FPr和酶IIIfru活性分布于这两个部分之间。用丁醇和尿素提取膜导致与膜相关的酶IIIfru和FPr活性定量释放，而酶IIfru仍与膜结合。使用离子交换色谱、凝胶过滤和反相高压液相色谱（HPLC）将FPr纯化至同质，并测定了其氨基酸组成和N端序列。还使用离子交换色谱、凝胶过滤和羟基磷灰石柱色谱将FPr和酶IIIfru复合物（Mr 50,000）纯化至接近同质。当通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析纯化的复合物时，它显示为两条蛋白带，迁移率对应于分子量约为40,000（酶IIIfru）和9,000（FPr）。在上述色谱步骤中或使用包括反相HPLC在内的其他几种技术时，FPr和酶IIIfru活性以及由这两条带代表的蛋白质均未分离，表明它们之间的结合非常紧密。从未分离或观察到不含FPr的活性酶IIIfru。在盐酸胍或尿素存在下，通过凝胶过滤可以将蛋白质以变性形式分离。对游离FPr和FPr-酶IIIfru复合物进行了表征，并将游离和复合FPr的性质与HPr的性质进行了比较。
