Conformational changes in glutamine synthetase from Escherichia coli. I. The binding of Mn2+ in relation to some aspects of the enzyme structure and activity.
Conformational changes in glutamine synthetase from Escherichia coli. I. The binding of Mn2+ in relation to some aspects of the enzyme structure and activity.
复制标题
大肠杆菌谷氨酰胺合成酶的构象变化。
DOI：
10.1021/bi00832a055
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发表时间：
1969
期刊：
影响因子：
2.9
通讯作者：
A. Ginsburg
中科院分区：
文献类型：
--
作者：
M. Denton;A. Ginsburg
材料与方法谷氨酰胺合成酶按Woolfolk等的方法纯化。（1966）从E.在谷氨酰胺合成酶制剂II所述的条件下生长的大肠杆菌细胞（Kingdon和Stadtman，1967; Shapiro等，1967年）。使用两种不同的酶制剂：例如，制备前描述的制剂II（Shapiro等人，1967），并且含有平均9当量的共价结合的AMP/摩尔酶（600，000g）; Ejl也被大规模分离，并且通过Shapiro等人概述的方法表征为含有平均2.3当量的AMP。（1967年）。包括E275的氨基酸分析（在密封管中在107下水解16、24和48小时），并且显示与文献I和II、E271和Eg的公开氨基酸组成分别没有显著偏差（Shapiro等人，1967），其中Eg水解产物作为对照进行分析。Eg3制剂腺苷酰化程度相对较低的原因尚不清楚。两个可能的原因是：细胞处于稳定生长期的时间（8 - 10批产生12.5 kg）发生变化;处理粗提物的时间较长（~60 l.）当去氧烯基化催化（Shapiro和Stadtman，1968）可能发生时。这些酶制剂的物理性质以及蛋白质浓度的分光光度法测定已在别处描述（Shapiro和Ginsburg，1968）。通过磷酸盐测定（Wool-folk和Stadtman，1964）或在23 - 24分光光度测定结合丙酮酸激酶和乳酸脱氢酶以测量ADP形成来测定酶活性（Kingdon等人，1968年）。在23 - 25 ° C下用配备有刻度扩展器和利兹和Northrup微电极（编号124138）的辐射计型PHM 25 pH计进行pH测定（并且如果需要，使用缓冲液的预定温度依赖性校正到实验温度），所述微电极具有低的KCl泄漏（Ginsburg和卡罗尔，1965）。S4MnCl 2（无载体）购自新英格兰核公司（Nuclear Corp.）。咪唑（Eastman Organic Chemicals）的储备溶液用木炭轻微处理并过滤以除去任何黄色; 2-甲基咪唑（Aldrich Chemical Co.）从去离子水中重结晶三次，然后用Chelex树脂（加州生物化学研究公司）进一步处理2-甲基咪唑的储备溶液以除去痕量的金属离子。所用无机盐为试剂级。用于酶测定的去离子水和材料描述于随附论文中（Ginsburg，1969）。平衡透析。如Ginsburg和Mehler（1966）所述，获得、制备和使用平衡透析的材料和技术，除非另有说明。在透析袋和烧瓶中，而不是在平衡透析池中，以低游离浓度的锰离子（54Mn2+）进行平衡。在54Mn 2+结合实验中，透析液缓冲液为0.1 m KCl和0.02 m氯化咪唑（pH <7.6）或0.1 m KCl和0.02 m氯化2-甲基咪唑（pH> 7.7）。酶溶液在pH 7.4（4）下用上述还含有5 µ MnClg的咪唑缓冲液预透析。用0.1mKCl、0.02m2-甲基咪唑氯化物和0.1mMnCl4（pH 8）平衡pH 8的酶溶液，通过G-25 Sephadex在25 ℃下凝胶过滤。然后将54Mn 2+添加到平衡的蛋白质溶液（~11mg/ml）中，并针对无Mn 2+的缓冲液（具有相同的盐组成）进行平衡。
Materials and MethodsGlutamine Synthetase was purified by the procedure of Woolfolk et al.(1966) from E. coliW cells, grown under the conditions described for the glutamine synthetase preparation II (Kingdon and Stadtman, 1967; Shapiro et al., 1967). Two different enzyme prepara-tions were used: Eg is preparation II described pre-viously (Shapiro et al., 1967) and contains an average of 9 equiv of covalently bound AMP/mole of enzyme (600,000 g); Ejl was isolated also on a large scale and it was characterized as containing an average of 2.3 equiv of AMP by the procedures outlined by Shapiro et al.(1967). Amino acid analysis of E275 (hydrolyzed in sealed tubes at 107 for 16, 24, and 48 hr) were included and showed no significant deviations from the published amino acid composition of prepara-tions I and II, E^ i and Eg, respectively (Shapiro et al., 1967) with Eg hydrolysates analyzed as controls. The reason for the relativelylow degree of adenylyla-tion of the Eg3 preparation is unclear. Two possible reasons are: variations occurred in the time that the cells (8-10 batches yielding 12.5 kg) were in the sta-tionary growth phase; the time was longer in handling the crude extract (~ 60 1.) when deadenylylating catalysis (Shapiro and Stadtman, 1968) could occur. The physical properties of these enzyme preparations as well as the spectrophotometric determination of protein concentrations have been described elsewhere (Shapiro and Ginsburg, 1968). Enzymatic activities were determined by either the phosphate assay (Wool-folk and Stadtman, 1964) or the spectrophotometric assay at 23-24 coupled with pyruvate kinase and lactic dehydrogenase to measure ADP formation (Kingdon et al., 1968). pH determinations were made at 23-25 (and corrected if necessary to thetemperature of the experiment using the predetermined temperature dependence of the buffer) with a Radiometer type PHM25 pH meter equipped with a scale expander and with the Leeds and Northrup microelectrodes (no. 124138) which have a low leakage of KC1 (Ginsburg and Carroll, 1965).Reagents. S4MnCl2 (carrier-free) was purchased from the New England Nuclear Corp. Stock solutions of imidazole (Eastman Organic Chemicals) were treated lightly with charcoal and filtered to remove any yellow color; 2-methylimidazole (Aldrich Chemical Co.) was recrystallized three times from deionized water and then stock solutions of 2-methylimidazole were further treated with Chelex resin (California Corp. for Biochemical Research) to remove traces of metal ions. Inorganic saltsused were reagent grade. The deionized water and the materials used for the enzyme assays are described in an accompanying paper (Ginsburg, 1969). Equilibrium Dialysis. Materials and techniques for equilibrium dialysis were obtained, prepared, and used as noted by Ginsburg and Mehler (1966) unless other-wise noted. Equilibration with manganous ions (54Mn2+) at low free concentrations was carried out in dialysis bags and flasks rather than in equilibrium dialysis cells. The dialysate buffers were either 0.1 m KC1 and 0.02 m imidazole chloride (pH< 7.6) or 0.1 m KC1 and 0.02 m 2-methylimidazole chloride (pH> 7.7) in the 54Mn2+ binding experiments. Enzyme solutions were predialyzed at pH 7.4 (4) against the above imidazole buffer containing also 5 µ MnClg. Theenzyme solutions at pH 8 were equilibrated with 0.1 m KC1, 0.02 m 2-methylimidazole chloride, and 0.1 mM MnCU (pH 8) by gel filtration at 25 through G-25 Sephadex. 54Mn2+ was added then to the equilibrated protein solutions (~ 11mg/ml), and equilibration against a Mn2+-free buffer (with an otherwise identical salt composition) was …