Sommer P, Klein J P, Schöller M, Frank R M
Infect Immun. 1985 Feb;47(2):489-95. doi: 10.1128/iai.47.2.489-495.1985.
A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.
从变形链球菌OMZ175中纯化出一种细胞质中的依赖果糖-1,6-二磷酸的乳酸脱氢酶（LDH；EC 1.1.1.27），经十二烷基硫酸钠-凝胶电泳判断已达到均一性。纯化过程包括对细胞质部分进行硫酸铵沉淀、DEAE-琼脂糖凝胶和蓝色琼脂糖凝胶CL.6B柱层析以及Sephacryl S200凝胶渗透。纯化酶的催化活性需要果糖-1,6-二磷酸的存在，在pH 5至6.2之间有较宽的最适范围。达到最大速度一半时所需的果糖-1,6-二磷酸浓度约为0.02 mM，并受丙酮酸浓度的影响。该酶似乎至少有两个与激活剂结合的位点，它们以协同方式相互作用。果糖-1,6-二磷酸浓度增加至2 mM可增强酶对丙酮酸的相对亲和力，并将丙酮酸饱和曲线从S形改变为双曲线形。酶活性对NADH也呈S形响应，显示出该辅因子有两个结合位点，希尔系数约为1.9。通过在Sephacryl S200上进行凝胶渗透测定，天然酶的分子量为150,000。经十二烷基硫酸钠-凝胶电泳观察到单体（38,000道尔顿）和二聚体（85,000道尔顿）；用2-巯基乙醇还原后，后一种形式会解离，该酶可被认为是四聚体。针对纯化的变形链球菌OMZ175 LDH获得的抗体与来自不同变形链球菌血清型、血链球菌OMZ9、干酪乳杆菌ATCC 4646和黏性放线菌NY 1的十二烷基硫酸钠解离形式的LDH发生交叉反应。竞争性酶联免疫吸附测定使我们能够检测到变形链球菌血清型和血链球菌的L-LDH天然状态之间非常密切的关系。还观察到与黏性放线菌和干酪乳杆菌的LDH有交叉反应，后者的相关性最小。在变形链球菌OMZ175的L-LDH和莱氏乳杆菌的D-LDH之间获得了非常弱的免疫关系，而与哺乳动物LDH未检测到交叉反应。
