Maret W
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):237-41. doi: 10.1073/pnas.91.1.237.
Mammalian metallothionein has been postulated to play a pivotal role in cellular zinc distribution. All seven of its metal atoms are bound with high thermodynamic stability in two clusters buried deeply in the molecule. If the protein is to function in metal delivery, there must be a biological mechanism to facilitate metal release. One means to achieve this would be a labilization of the clusters by interaction of metallothionein with an appropriate cellular ligand. To search for such a mediator, we have designed a rapid radiochromatographic method that can detect changes in the zinc content of 65Zn-labeled metallothionein in response to other biomolecules. Using this methodology, we have established that rabbit liver metallothionein 2 interacts with glutathione disulfide with concomitant release of zinc. Under conditions of pseudo-first-order kinetics, the monophasic reaction depends linearly on the concentration of glutathione disulfide in the range from 5 to 30 mM with a second-order rate constant k = 4.9 x 10(-3)s-1.M-1 (pH 8.6; 25 degrees C). Apparently, zinc release does not involve direct access of glutathione disulfide to the inner coordination sphere of the metals. Rather it appears that the solvent-accessible zinc-bound thiolates in two clefts of each domain of metallothionein [Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F., Wang, B. C. & Stout, C. D. (1991) J. Mol. Biol. 221, 1269-1293] participate in a thiol/disulfide interchange with glutathione disulfide. This rate-limiting initial S-thiolation, which occurs with indistinguishable rates in both clusters, then causes the clusters to collapse and release their zinc. Such a mechanism of metal release would link the control of the metal content of metallothionein to the cellular glutathione redox status and raises important questions about the physiological implications of this observation with regard to a role of glutathione in zinc metabolism and in making zinc available for other biomolecules.
哺乳动物金属硫蛋白被认为在细胞锌分布中起关键作用。其所有七个金属原子以高热力学稳定性结合在分子内部深处的两个簇中。如果该蛋白质要在金属传递中发挥作用，那么必须存在促进金属释放的生物学机制。实现这一点的一种方法是通过金属硫蛋白与合适的细胞配体相互作用使簇不稳定。为了寻找这样的介质，我们设计了一种快速放射色谱方法，该方法可以检测响应其他生物分子时，65Zn标记的金属硫蛋白锌含量的变化。使用这种方法，我们确定兔肝金属硫蛋白2与谷胱甘肽二硫化物相互作用并伴随锌的释放。在准一级动力学条件下，单相反应在5至30 mM范围内线性依赖于谷胱甘肽二硫化物的浓度，二级速率常数k = 4.9 x 10(-3)s-1.M-1（pH 8.6；25℃）。显然，锌的释放并不涉及谷胱甘肽二硫化物直接进入金属的内配位球。相反，似乎在金属硫蛋白每个结构域的两个裂隙中可被溶剂接触的锌结合硫醇盐[罗宾斯，A. H.，麦克里，D. E.，威廉姆森，M.，科利特，S. A.，徐昂，N. H.，富雷，W. F.，王，B. C. & 斯托特，C. D.（1991）J. Mol. Biol. 221，1269 - 1293]参与了与谷胱甘肽二硫化物的硫醇/二硫化物交换。这种限速的初始S - 硫醇化在两个簇中以难以区分的速率发生，然后导致簇塌陷并释放它们的锌。这种金属释放机制将金属硫蛋白的金属含量控制与细胞谷胱甘肽氧化还原状态联系起来，并就谷胱甘肽在锌代谢中的作用以及使锌可用于其他生物分子这一观察结果的生理意义提出了重要问题。
