Vadeboncoeur C, Proulx M, Trahan L
Can J Microbiol. 1983 Dec;29(12):1694-705. doi: 10.1139/m83-260.
The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.
糖磷酸转移酶系统（PTS）由几种蛋白质组成。其中两种被指定为通用蛋白，因为它们是PTS所有糖类运输和磷酸化所必需的。这两种蛋白质存在于细胞提取物的可溶部分，分别称为HPr和酶I（EI）。我们在这项工作中报道了从唾液链球菌ATCC 25975中纯化和鉴定这两种蛋白质的过程。HPr通过DEAE-纤维素色谱、Ultrogel AcA44分子筛和羧甲基纤维素色谱进行纯化。在尿素存在下的十二烷基硫酸钠电泳显示出一条分子量为6700的单一带。该蛋白质不含色氨酸，pI为4.8。EI的纯化方案如下：DEAE-纤维素色谱、羟基磷灰石色谱、DEAE-葡聚糖A-50色谱、制备电泳和Ultrogel AcA34分子筛。EI的五步纯化产生了199倍纯化的制剂，在37℃下的比活性为每毫克蛋白质每分钟磷酸化530μmol HPr。在Ultrogel AcA34上过滤后得到的级分在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上给出一条带（68000）。在4℃下通过凝胶过滤测定的天然酶分子量为135000，表明它是二聚体。酶I的pI为4.2，最适pH为6.7，对HPr的Km约为27μM，对磷酸烯醇丙酮酸的Km为0.48 mM，其动力学符合乒乓机制。已获得证据表明，唾液链球菌酶I与变形链球菌各菌株的酶I在抗原性上非常相似，但与枯草芽孢杆菌、金黄色葡萄球菌、粪肠球菌和大肠杆菌的酶不同。
