Improved Aequorin for Ca Assay in Functioning Cells

发光蛋白水母发光蛋白（最初由P.I.在 1962）已被广泛用作活细胞中的Ca -指示剂。 然而，过去使用的水母发光蛋白的制剂是 含有杂质的不确定的异水母发光蛋白混合物。 最近，在实验室中制备了两种改进类型的水母发光蛋白。 P.I.的实验室：第一种是纯异水母发光蛋白， 第一种是单分子物种，第二种是半合成的 称为e-水母发光蛋白的光蛋白，其中官能团 被修改了。 e-aequorin的发光光谱显示两个 峰（405 nm和465 nm）和峰强度的比值 是卡！-依赖于5 - 7的pCa范围，从而允许 测定钙 浓度直接从强度 两个峰值的比值。 该项目的主要目标是建立 在测量中使用异水母发光蛋白和e-水母发光蛋白的优点 细胞内钙，与其他钙指标相比;这 将通过实际使用这些材料来实现， 合作的细胞生物学家 足够量的 将为此目的生产异水母发光蛋白和e-水母发光蛋白 并遵守其他调查人员的要求。 到 增加更有用类型的异水母发光蛋白的产量， 将研究异水母发光蛋白之间的相互转化。 的 第二个目的是制备其它有用形式的半- 合成水母发光蛋白，其优于异水母发光蛋白和e-水母发光蛋白;该 将通过进一步修饰官能团来完成。

The photoprotein aequorin (initially isolated by the P.I. in 1962) has been widely used as a Ca -indicator in living cells. However, the preparations of aequorin used in the past were undefinable mixtures of isoaequorins containing impurities. Recently, two improved types of aequorin were prepared in the P.I.'s laboratory: the first is pure isoaequorins consisting of single molecular species, and the second is a semi-synthetic photoprotein designated e-aequorin in which the functional group is modified. The luminescence spectrum of e-aequorin shows two peaks (405 nm and 465 nm) and the ratio of the peak intensities is ıCa !-dependent in the pCa range of 5 - 7, thus allowing the determination of Ca concentration directly from the intensity ratio of the two peaks. The primary objective of this project is to establish the advantage of using isoaequorins and e-aequorin in measuring intracellular Ca , compared with other Ca -indicators; this will be accomplished by the actual use of those materials by collaborating cell biologists. Sufficient amounts of isoaequorins and e-aeqourin will be produced for this purpose and also to comply with requests from other investigators. To increase the yields of more useful types of isoaequorins, interconversion between isoaequorins will be investigated. The second objective is to prepare other useful forms of semi- synthetic aequorin which excel isoaequorins and e-aequorin; this will be done by further modification of the functional group.