Mezna M, Michelangeli F
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):177-82. doi: 10.1042/bj3250177.
Thimerosal, a thiol-reactive reagent, has been shown to increase the cytosolic Ca2+ concentration in a variety of cells by sensitizing inositol 1,4,5-trisphosphate (InsP3) receptors. Thimerosal can have both sensitizing (at concentrations of <2 microM) and inhibitory (at concentrations of >2 microM) effects on InsP3-induced Ca2+ release (IICR) from cerebellar microsomes. Transient kinetic studies were performed by employing a fluorimetric stopped-flow approach using fluo-3. IICR was found to be a bi-exponential process with a fast and a slow component. At a maximal InsP3 concentration (20 microM), the fast phase had a rate constant of 0.9 s-1 and the slow phase had a rate constant of 0.4 s-1. The amplitudes of the two phases were 60% and 40% respectively. When the rate constants for the two phases were plotted as Hill plots, the processes were found to be non-co-operative in both cases (Hill coefficient of 1.0), thus arguing for a simple mechanism linking InsP3 binding to channel opening. At a submaximal InsP3 concentration (0.2 microM), where the sensitizing effects of thimerosal are most pronounced, thimerosal increased the rate constants of both phases in a sigmoidal fashion, with a Hill coefficient of 4.0, suggesting that several cysteine residues (up to four) need to be modified in order for maximum sensitization to occur. The rate constants remained elevated even at thimerosal concentrations that inhibited IICR. The amplitude or extent of Ca2+ release was, however, elevated to a much greater extent in the slow phase, suggesting that the two phases respond differently. At maximal InsP3 concentrations, thimerosal has no effect upon the rate constants but inhibits the amplitude of Ca2+ release.
硫柳汞是一种硫醇反应试剂，已证明它可通过使肌醇1,4,5 - 三磷酸（InsP3）受体敏感化来增加多种细胞胞质中的Ca2+浓度。硫柳汞对小脑微粒体中InsP3诱导的Ca2+释放（IICR）可产生敏感化作用（浓度<2 microM时）和抑制作用（浓度>2 microM时）。采用荧光停流法结合fluo - 3进行了瞬态动力学研究。发现IICR是一个具有快速和慢速成分的双指数过程。在最大InsP3浓度（20 microM）下，快速相的速率常数为0.9 s-1，慢速相的速率常数为0.4 s-1。两个相的幅度分别为60%和40%。当将两个相的速率常数绘制为希尔图时，发现这两个过程在两种情况下均为非协同性（希尔系数为1.0），因此表明存在一种将InsP3结合与通道开放联系起来的简单机制。在亚最大InsP3浓度（0.2 microM）下，硫柳汞的敏感化作用最为明显，硫柳汞以S形方式增加了两个相的速率常数，希尔系数为4.0，这表明需要修饰几个半胱氨酸残基（最多四个）才能实现最大程度的敏感化。即使在抑制IICR的硫柳汞浓度下，速率常数仍保持升高。然而，Ca2+释放的幅度或程度在慢速相中升高的幅度要大得多，这表明两个相对硫柳汞的反应不同。在最大InsP3浓度下，硫柳汞对速率常数没有影响，但会抑制Ca2+释放的幅度。
