FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima -: Quantitation, GTP hydrolysis, and assembly
FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima -: Quantitation, GTP hydrolysis, and assembly
复制标题
DOI：
10.1002/(sici)1097-0169(1998)40:1
复制
发表时间：
1998-01-01
影响因子：
--
通讯作者：
Erickson, HP
中科院分区：
文献类型：
--
作者：
Lu, CL;Stricker, J;Erickson, HP
我们从海栖热袍菌和维氏固氮菌中克隆了 ftsZ 基因，并在大肠杆菌中表达了蛋白（TmFtsZ 和 AzFtsZ）。我们将这些蛋白质与大肠杆菌 FtsZ (EcFtsZ) 进行比较，发现所有三个物种的 GTPase 活性的几个显着特征都是相似的，这意味着这些特征在 FtsZ 中可能是普遍存在的。使用校准的蛋白质测定，我们发现所有三个 FtsZ 每摩尔 FtsZ 结合 1 摩尔鸟嘌呤核苷酸，并以高速率水解 GTP（每 FtsZ 每分钟 > 2 GTP）。所有三种物质都需要镁和一价阳离子来进行 GTP 水解。之前的报告表明，EcFtsZ（某些其他物种）需要钾。我们证实了 EcFtsZ 的这种特异性，但发现钾和钠都对 Az-和 TmFtsZ 起作用。特异性 GTP 酶活性对 FtsZ 浓度具有显着依赖性：活性（每个 FtsZ 分子）不存在或低于 50 μg/ml，从 50 急剧上升至 300 μg/ml，并稳定在高于 300 μg/ml 的恒定高值。这一发现表明活性状态需要以 50-300 μg/ml 协同组装的聚合物。通过负染色电子显微镜观察活性聚合物的良好候选物——在具有活性GTP酶的所有条件下都可以看到直的原丝和原丝对。我们认为 FtsZ 的 GTP 水解可能与组装耦合，就像微管蛋白一样，在 FtsZ 单体结合到原丝末端后不久就会发生水解。作为本研究的一部分，我们通过定量氨基酸分析测定了 EcFtsZ 和 TmFtsZ 的浓度，并用它来标准化二金鸡宁酸比色测定。这是首次准确测定 FtsZ 浓度。使用这种标准的定量蛋白质印迹法，我们确定每个大肠杆菌细胞平均含有 15,000 个 FtsZ 分子，浓度为 400 μg/ml。这刚好高于体外完整 GTP 酶活性的平台值。 (C) 1998 Wiiey-Liss, Inc.
We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli. We compared these proteins to E. coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs. Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (>2 GTP per FtsZ per min). All three required magnesium and a monovalent cation for GTP hydrolysis. Previous reports showed that EcFtsZ land some other species) required potassium. We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az-and TmFtsZ. Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 mu g/ml, rose steeply from 50 to 300 mu g/ml and plateaued at a constant high value above 300 mu g/ml. This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 mu g/ml. A good candidate for the active polymer was visualized by negative stain electron microscopy-straight protofilaments and protofilament pairs were seen under all conditions with active GTPase. We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end. As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay. This is the first accurate determination of FtsZ concentration. Using this standard and quantitative Western blotting, we determined that the average E. coli cell has 15,000 molecules of FtsZ, at a concentration of 400 mu g/ml. This is just above the plateau for full GTPase activity in vitro. (C) 1998 Wiiey-Liss, Inc.