Dityrosine formation in calmodulin: cross-linking and polymerization catalyzed by Arthromyces peroxidase.
Dityrosine formation in calmodulin: cross-linking and polymerization catalyzed by Arthromyces peroxidase.
复制标题
钙调蛋白中二酪氨酸的形成：节霉菌过氧化物酶催化的交联和聚合。
DOI：
10.1021/bi9526037
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
Anderson,SR
中科院分区：
文献类型：
--
作者：
Malencik,DA;Anderson,SR
我们使用牛脑钙调蛋白，一种受光活化二硫酪氨酸形成的蛋白质[Malencik，D.A.，&Anderson，S.R.(1987)BioChemical 26,695；(1994)BioChemical 33,13363]作为开发有效的酶催化蛋白质交联技术的模型。制定该程序的关键步骤是(1)鉴定来自Arthromycesramosus的过氧化物酶，它催化蛋白质中不被其他常见过氧化物酶作用的Dityroine的产生，(2)监测Dityroine的本征荧光，以确定最佳反应条件，在含有硼酸−硼酸钠(浓度≥0.2M)、∼pH 8.3、∼40°C的钙调蛋白溶液中实现，以及(3)与还原型谷胱甘肽的反应猝灭。Arthromycesperase是唯一能够通过主要是分子间反应催化CaM中Dityroine产生的常见过氧化物酶。凝胶过滤产生的组分(占初始钙调素的∼的40%)在NaDodSO4聚丙烯酰胺凝胶电泳法中表现出不同的迁移率范围，并含有接近可能的最大二酪氨酸量。不同组分经历了在沉积速度和/或荧光各向异性测量中检测到的依赖于钙的构象变化。大多数样品都能刺激肌球蛋白轻链激酶的钙依赖活性。在使用人工合成的磷酸受体多肽的催化分析中，其平均活性从天然钙调蛋白的50%到100%不等。然而，只有最少的聚合部分和光产生的钙调蛋白二聚体显著提高了钙调神经磷酸酶的活性。能够制备保持相当程度的生物活性并显示出双酪氨酸的强烈可见荧光的可溶性钙调蛋白聚合物的能力表明了关节霉菌过氧化物酶在蛋白质的零长度交联中的潜在用途。
We employ bovine brain calmodulin, a protein that is subject to photoactivated dityrosine formation [Malencik, D. A., & Anderson, S. R. (1987)Biochemistry26, 695; (1994)Biochemistry33, 13363], as a model for the development of an efficient enzyme-catalyzed protein cross-linking technique. Key steps in the elaboration of the procedure are (1) identification of a peroxidase, fromArthromyces ramosus, that catalyzes dityrosine production in proteins that are not acted on by other common peroxidases, (2) monitoring of the intrinsic fluorescence of dityrosine to determine optimum reaction conditions, achieved with calmodulin in solutions containing boric acid−sodium borate (concentration ≥0.2 M), ∼pH 8.3, ∼40 °C, and (3) quenching of the reaction with reduced glutathione.Arthromycesperoxidase is the only common peroxidase able to catalyze significant dityrosine production in calmodulin, through a reaction that is largely intermolecular. Gel filtration yields fractions (accounting for ∼40% of the initial calmodulin) that represent differing mobility ranges in NaDodSO4polyacrylamide gel electrophoresis and contain close to the maximum possible amounts of dityrosine. The various fractions undergo Ca2+-dependent conformational changes detected in sedimentation velocity and/or fluorescence anisotropy measurements. Most of the samples stimulate the Ca2+-dependent activity of smooth muscle myosin light chain kinase. In catalytic assays utilizing the synthetic phosphate acceptor peptide, the average activities range from 50 to 100% of that determined for native calmodulin. However, only the least polymerized fraction and the photogenerated calmodulin dimers significantly enhance thep-nitrophenylphosphatase activity of calcineurin. The ability to prepare soluble calmodulin polymers that retain a substantial degree of biological activity and exhibit the intense visible fluorescence of dityrosine illustrates the potential usefulness ofArthromycesperoxidase in the zero-length cross-linking of proteins.