钙调蛋白中双酪氨酸的形成。

Dityrosine formation in calmodulin.

作者信息

Malencik D A, Anderson S R

出版信息

Biochemistry. 1987 Feb 10;26(3):695-704. doi: 10.1021/bi00377a006.

Abstract

Ultraviolet (280-nm) irradiation of bovine brain calmodulin results in calcium-dependent changes in its fluorescence emission spectrum. These consist of a decline in the intrinsic tyrosine fluorescence of the protein and the appearance of a new emission maximum at 400 nm. Chromatography of irradiated calmodulin, using Ultrogel AcA 54 and phenyl-agarose columns, yields several distinctive fractions. One of these, representing 2.8% of the total recovered protein and 53% of the total fluorescence emission at 400 nm, was selected for detailed characterization. Analyses performed on acid hydrolysates reveal the presence of dityrosine, a derivative of tyrosine known for its fluorescence near 400 nm, at the level of 0.59-0.89 mol per 16,700 g of protein. Sodium dodecyl sulfate gel electrophoresis experiments demonstrate two components of apparent molecular weights 14,000 (80%) and 16,000 (20%). Observations on the effects of UV irradiation on the thrombic fragments of calmodulin and on related calcium binding proteins (rabbit skeletal muscle troponin C, bovine cardiac troponin C, and parvalbumin) support the interpretation that dityrosine formation in calmodulin results from the intramolecular cross-linking of Tyr-99 and Tyr-138. The dityrosine-containing photoproduct of calmodulin is unable to stimulate the p-nitrophenyl phosphatase activity of calcineurin under standard assay conditions. Fluorescence titrations show a generally weakened interaction with calcium ion occurring in two stages. The pKa of the derivative is considerably higher than that of free dityrosine and is calcium dependent, decreasing from 7.88 to 7.59 on the addition of 3 mM CaCl2. Smooth muscle myosin light chain kinase binds the derivative about 280-fold less effectively than it binds native calmodulin. Of several metal ions tested, only Cd2+ approaches Ca2+ in its ability to promote the appearance of the 400-nm emission band during UV irradiation of calmodulin. Mn2+ and Cu2+ appear to inhibit dityrosine formation. Ascorbic acid, dithiothreitol, and glutathione are also inhibitory.

摘要

用紫外线（280纳米）照射牛脑钙调蛋白会导致其荧光发射光谱发生钙依赖性变化。这些变化包括蛋白质固有酪氨酸荧光的下降以及在400纳米处出现新的发射最大值。使用Ultrogel AcA 54和苯基琼脂糖柱对辐照后的钙调蛋白进行色谱分析，可得到几个不同的组分。其中一个组分占回收总蛋白的2.8%，在400纳米处的总荧光发射占53%，被选出来进行详细表征。对酸水解产物的分析表明，每16700克蛋白质中存在0.59 - 0.89摩尔的二酪氨酸，二酪氨酸是酪氨酸的一种衍生物，在400纳米附近有荧光。十二烷基硫酸钠凝胶电泳实验表明有两种表观分子量分别为14000（80%）和16000（20%）的组分。对紫外线照射钙调蛋白的血栓片段以及相关钙结合蛋白（兔骨骼肌肌钙蛋白C、牛心肌肌钙蛋白C和小清蛋白）的影响的观察结果支持了这样的解释，即钙调蛋白中二酪氨酸的形成是由Tyr - 99和Tyr - 138的分子内交联导致的。在标准测定条件下，含二酪氨酸的钙调蛋白光产物无法刺激钙调神经磷酸酶的对硝基苯磷酸酶活性。荧光滴定显示与钙离子的相互作用通常在两个阶段减弱。该衍生物的pKa值明显高于游离二酪氨酸的pKa值，且依赖于钙，加入3 mM氯化钙后从7.88降至7.59。平滑肌肌球蛋白轻链激酶结合该衍生物的效率比结合天然钙调蛋白低约280倍。在测试的几种金属离子中，只有Cd2+在紫外线照射钙调蛋白期间促进400纳米发射带出现的能力接近Ca2+。Mn2+和Cu2+似乎抑制二酪氨酸的形成。抗坏血酸、二硫苏糖醇和谷胱甘肽也有抑制作用。