Gibson J L, Tabita F R
J Bacteriol. 1987 Aug;169(8):3685-90. doi: 10.1128/jb.169.8.3685-3690.1987.
A heterologous phosphoribulokinase (PRK) gene probe was used to analyze two recombinant plasmids isolated from a Rhodopseudomonas (Rhodobacter) sphaeroides gene library. These plasmids were previously shown to carry the genes for form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O). Southern blot hybridization analysis indicated that there were two PRK genes linked to the RuBPC/O coding sequences. Restriction mapping showed the arrangement of the duplicate sets of PRK and RuBPC/O to be distinct. Subcloning of the hybridizing PRK sequences downstream of the lac promoter of pUC8 allowed expression of the two PRK enzymes in Escherichia coli. Analysis of the purified proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed polypeptides with molecular weights of 32,000 and 34,000 corresponding to the form I and form II PRKs, respectively. Preliminary experiments on sensitivity to NADH regulation suggested that the two PRK enzymes differ in catalytic properties.
使用异源磷酸核酮糖激酶（PRK）基因探针分析从球形红假单胞菌（红杆菌属）基因文库中分离出的两个重组质粒。先前已表明这些质粒携带I型和II型核酮糖1,5-二磷酸羧化酶/加氧酶（RuBPC/O）的基因。Southern印迹杂交分析表明，有两个PRK基因与RuBPC/O编码序列相连。限制性图谱显示PRK和RuBPC/O重复序列的排列不同。将杂交的PRK序列亚克隆到pUC8的lac启动子下游，可使两种PRK酶在大肠杆菌中表达。通过十二烷基硫酸钠平板凝胶电泳对纯化蛋白进行分析，结果显示分子量分别为32,000和34,000的多肽，分别对应I型和II型PRK。对NADH调节敏感性的初步实验表明，这两种PRK酶的催化特性有所不同。
