Lahti R, Niemi T
J Biochem. 1981 Jul;90(1):79-85. doi: 10.1093/oxfordjournals.jbchem.a133471.
Inorganic pyrophosphatase [EC 3.6.1.1] from Streptococcus faecalis ATCC 8043 was purified to homogeneity as judged by slab gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The purification consisted of the following steps: streptomycin sulfate precipitation, (NH4)2SO4 treatment in a Sepharose CL-4B column, DEAE-Sepharose CL-6B chromatography, gel filtration on Ultrogel AcA 34, and preparative slab gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight and slab gel electrophoresis in the presence of sodium dodecyl sulfate to study the subunit molecular weight. The enzyme appeared to be composed of four subunits with molecular weights of approximately 32,500. The molecular weight of the native enzyme was about 128,000. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate, and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Ca2+ supported 24% and 5.6% of the activity observed with Mg2+, respectively. 2,4,6-Trinitrobenzene sulfonic acid inhibited the reductants containing SH-groups activated the enzyme suggesting that lysine and cysteine have essential roles in the enzyme.
从粪链球菌ATCC 8043中提取的无机焦磷酸酶[EC 3.6.1.1]，经十二烷基硫酸钠存在和不存在时的平板凝胶电泳判断，已纯化至同质。纯化过程包括以下步骤：硫酸链霉素沉淀、在琼脂糖CL-4B柱中进行硫酸铵处理、DEAE-琼脂糖CL-6B柱层析、在Ultrogel AcA 34上进行凝胶过滤以及制备性平板凝胶电泳。在Ultrogel AcA 34上进行凝胶过滤用于测定分子量，在十二烷基硫酸钠存在下进行平板凝胶电泳以研究亚基分子量。该酶似乎由四个亚基组成，分子量约为32,500。天然酶的分子量约为128,000。该酶催化无机焦磷酸的水解，对多种其他磷酸酯无活性。阳离子Mg2+是最大活性所必需的；Co2+和Ca2+分别支持观察到的Mg2+活性的24%和5.6%。2,4,6-三硝基苯磺酸抑制该酶，含SH基团的还原剂激活该酶，表明赖氨酸和半胱氨酸在该酶中起重要作用。
