ASSOCIATION DISSOCIATION OF MAMMALIAN BRAIN GLUTAMINE-SYNTHETASE - EFFECTS OF METAL-IONS AND OTHER LIGANDS
ASSOCIATION DISSOCIATION OF MAMMALIAN BRAIN GLUTAMINE-SYNTHETASE - EFFECTS OF METAL-IONS AND OTHER LIGANDS
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DOI：
10.1016/0003-9861(84)90559-9
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发表时间：
1984-01-01
影响因子：
3.9
通讯作者：
WEDLER, FC
中科院分区：
文献类型：
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作者：
DENMAN, RB;WEDLER, FC
羊脑谷氨酰胺合成酶在体内和体外均以Mn4E络合物形式存在，其中E为八聚体酶。以前观察到的添加金属离子和蛋白质浓度对体外观察到的比活性的异常影响现在可以用天然八聚体的缔合-解离来解释。在没有甘油的情况下，添加甘油以稳定酶的长期储存，由于八聚体解离为四聚体，进而解离为非活性单体，测定混合物中的活性急剧下降至4µg/ml(20 mM八聚体)以下。在任何条件下都没有检测到二聚体物种。八聚体Mn4EMn4可被Mn(II)进一步激活，形成比活力约为900U/mg的Mn4EMn4Mn8。含一个Mn(II)/亚基的酶Mn4EMn4与八聚体的结合比与Mn4E的结合更广泛。在以四聚体为主的低浓度酶作用下，单独或成对加入底物可引起八聚体的部分再结合，最有效的组合是三磷酸腺苷和谷氨酸，二磷酸腺苷和L-谷氨酰胺，或三磷酸腺苷和L-蛋氨酸亚磺胺。用库尔加诺夫或托姆斯方法分析数据表明，四聚体/八聚体平衡的Kd值约为2.5倍。10-6M，与其他酶系统的计算值相当。在镁(II)依赖的转移酶实验中，计算出八聚体和单体的比活为200。20U/mg和0U/mg。在分析条件下，直接测定纯四聚体的比活性会受到底物促进的重结合成八聚体的阻碍。由2M尿素产生的四聚体，然后固定在CNBr活化的Sepharose4B上，其比活力是相同处理的八聚体的86%。这表明比活度约为172(.+-。20)对于溶液中的四聚体。光散射实验表明，当每个亚基结合1.7-2.0个Mn(II)时，八聚体酶八聚体可以进一步结合到一个寡聚物种(Mn4EMn4Mn8)n，其中hivin.n.gtoreq。5.La~(3+)对齐聚反应也有较强的促进作用。镁(II)只引起四聚体与八聚体的缔合。对四聚体内亚单位结构域(假设亚基相同)、八聚体中四聚体之间以及八聚体之间相互作用的各种立体化学模型的分析表明，这些数据与产生八聚体的同源相互作用最一致，而不是异源相互作用。这些分析还预测，立方八聚体通过较弱的依赖于Mn(II)的相互作用形成低聚物也最有可能通过同源域发生。现有的新兴市场证据支持这些假设模型。四聚体内部的相互作用强于四聚体之间的相互作用，而四聚体之间的相互作用又强于八聚体之间的相互作用。
Glutamine synthetase from ovine brain exists in vivo and in vitro as a Mn4E complex, where E is octameric enzyme. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 .mu.g/ml (20 mM octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn4EMn4 could be activated further by Mn(II) to form a species Mn4EMn4Mn8 that has a specific activity of about 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn4EMn4, associated to octamers more extensively than Mn4E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and L-glutamine, or ATP and L-methionine sulfoximine. Analysis of the data by the Kurganov or Thomes methods indicates that the tetramer/octamer equilibrium had a Kd value of about 2.5 .times. 10-6 M, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 .+-. 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 M urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of about 172 (.+-. 20) for tetramers in solution. Light-scattering experiments showed that, with 1.7-2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn4EMn4Mn8)n, where .hivin.n .gtoreq. 5. Oligomerization also was promoted strongly by La3+. Mg(II) caused only the association of tetramer to octamer. Analysis of various stereochemical models for the interaction of subunit domains (assuming identical subunits) within tetramers, between tetramers in the octamers, and between octamers indicate that the data are most consistent with isologous, rather than heterologous, interactions to produce octamer. These analyses also predict that formation of oligomers from cubic octamers through weaker, Mn(II)-dependent interactions also are most likely to occur via isologous domains. The available EM evidence supports these hypothetical models. Interactions within tetramers are stronger than those between tetramers, which are stronger than those between octamers.