Kemple M D, Lovejoy M L, Ray B D, Prendergast F G, Nageswara Rao B D
Department of Physics, Indiana University-Purdue University, Indianapolis 46205-2810.
Eur J Biochem. 1990 Jan 12;187(1):131-5. doi: 10.1111/j.1432-1033.1990.tb15286.x.
Cation binding at 5 degrees C by aequorin, a bioluminescent protein from the jellyfish Aequorea victoria, was examined by means of Mn(II) EPR. The bioluminescence of aequorin is triggered by Ca(II), as well as by trivalent lanthanides, and is inhibited by Mg(II) and Mn(II). Three EF-hand Ca(II)-binding domains have been identified in the aequorin amino acid sequence. In the work reported here, active native aequorin was found to have a single tight binding site for Mn(II) with an association constant of 0.566 microM-1. Ca(II) and La(III) competed for the Mn(II) site with association constants of 1.92 microM-1 and 1.38 microM-1, respectively. The affinity of Ca(II) and La(III) for their two other (presumed) sites on aequorin was an order of magnitude less than their affinity for the Mn(II) site. Mg(II) competed for the Mn(II) site as well but with a much smaller association constant of 0.0109 microM-1. Ca(II)-independent discharged aequorin did not bind Mn(II) to a significant degree. Conjectures on the location of the Mn(II) site in the aequorin amino acid sequence and on the relationship between the binding parameters of the cations and their influence on aequorin activity are given.
利用锰（II）电子顺磁共振（EPR）技术研究了来自维多利亚多管水母的生物发光蛋白水母发光蛋白在5摄氏度下与阳离子的结合情况。水母发光蛋白的生物发光由钙（II）以及三价镧系元素触发，并受到镁（II）和锰（II）的抑制。在水母发光蛋白的氨基酸序列中已鉴定出三个EF手型钙（II）结合结构域。在本文报道的工作中，发现活性天然水母发光蛋白对锰（II）有一个单一的紧密结合位点，其缔合常数为0.566 μM⁻¹。钙（II）和镧（III）分别以1.92 μM⁻¹和1.38 μM⁻¹的缔合常数竞争锰（II）位点。钙（II）和镧（III）对水母发光蛋白上另外两个（假定的）位点的亲和力比对锰（II）位点的亲和力小一个数量级。镁（II）也竞争锰（II）位点，但其缔合常数小得多，为0.0109 μM⁻¹。与钙（II）无关的已释放水母发光蛋白在很大程度上不结合锰（II）。文中给出了关于锰（II）位点在水母发光蛋白氨基酸序列中的位置以及阳离子结合参数与其对水母发光蛋白活性影响之间关系的推测。
