Katayama Y, Gottesman S, Pumphrey J, Rudikoff S, Clark W P, Maurizi M R
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1988 Oct 15;263(29):15226-36.
The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.
来自大肠杆菌的一种双组分、ATP 依赖性蛋白酶的 ATP 结合组分（组分 II，以下简称 ClpA）已被纯化至同质。ClpA 是一种亚基分子量为 81,000 的蛋白质。它具有内在的 ATP 酶活性，并且仅在第二种组分（组分 I，以下简称 ClpP）、Mg2+和 ATP 存在的情况下才激活蛋白质底物的降解。在不同培养基中以及在 30 至 42 摄氏度的温度下生长的细胞中，ClpA 的量变化不到 2 倍。ClpA 似乎不是一种热休克蛋白，因为它的合成不依赖于 htpR。针对纯化的 ClpA 的抗体被用于鉴定携带 clpA 基因的λ转导噬菌体。克隆的基因包含一个预期编码 ClpA 前 28 个氨基酸的 DNA 序列，这是通过对纯化的 ClpA 进行蛋白质测序确定的。噬菌体中的 clpA 基因通过插入δkan 缺陷转座子而发生突变，并且这些突变通过同源重组转移到大肠杆菌中。clpA 基因被定位到大肠杆菌染色体上的 19 分钟处。在基因早期有插入的突变细胞在 Western 印迹中检测不到 ClpA 蛋白，并且这种突变细胞的提取物没有可检测到的 ClpA 活性。clpA - 突变体在所有测试条件下生长良好，在氮饥饿期间蛋白质周转方面没有缺陷，在诸如λ蛋白 O、N 和 cII 或大肠杆菌蛋白 SulA、RcsA 和谷氨酸脱氢酶等高度不稳定蛋白质的周转方面也没有缺陷。clpA 突变体中含刀豆氨酸的异常蛋白质的降解存在缺陷，特别是在也有 lon - 突变的细胞中。clpA - lon - 细胞的提取物具有 ATP 依赖性酪蛋白降解活性。
