Presta A, Stillman M J
Department of Chemistry, University of Western Ontario, London, Canada.
J Inorg Biochem. 1997 Jun;66(4):231-40. doi: 10.1016/s0162-0134(96)00216-4.
Copper is an essential metal ion to many living organisms, including mammals, as it mediates a wide variety of important biochemical processes. At elevated concentrations, copper is extremely toxic to host cells. This paradoxical nature of copper has necessitated a highly regulated procedure for its cellular accumulation, transport, and excretion. One important group of proteins involved in eukaryotic copper speciation is the protein metallothionein. Luminescence microscopy data, emission, and circular dichroism spectral data are reported as copper is incorporated into metallothionein by the yeast Saccharomyces cerevisiae. These techniques provide information on the mechanism of copper uptake by S. cerevisiae. A two-stage kinetic mechanism for the uptake of copper from the growth medium by the yeast cells is observed. The first stage displays an uptake rate that is dependent on the initial copper concentration of the growth medium, and lasts for approximately 6 h. The second stage has a slower rate of copper uptake than the first, but the kinetics are independent of the initial copper concentration. Emission spectra recorded directly from the intact yeast cells (at 77 K) show that the cellular incorporation of copper proceeds via several species, eventually leading to storage of the copper in the form of Cu-metallothionein. The photomicrographs of yeast cells grown in a copper-containing medium clearly show an orange luminescence, indicating the formation of a Cu(I)-thiolate species. The identification of this species as copper-metallothionein was confirmed by measurement of the circular dichroism and emission properties following excretion and isolation of the copper-containing protein from the yeast cells. Analysis of the emission spectrum from S. cerevisiae Cu-metallothionein at 77 K reveals two emission bands, centered at 570 and 700 nm. The high-energy emission band exhibits a two-component decay, with excited state lifetimes of 4.70 and 48.5 microseconds. The low-energy emission exhibits one major decay component with a lifetime of 1.13 microseconds. A high-molecular-weight, copper-containing species is also isolated from the yeast cells and is characterized spectroscopically.
铜是包括哺乳动物在内的许多生物所必需的金属离子，因为它介导了多种重要的生化过程。在浓度升高时，铜对宿主细胞具有极高的毒性。铜的这种矛盾特性使得其细胞内积累、运输和排泄过程需要高度调控。参与真核生物铜形态形成的一类重要蛋白质是金属硫蛋白。本文报道了酿酒酵母将铜掺入金属硫蛋白过程中的发光显微镜数据、发射光谱和圆二色光谱数据。这些技术提供了关于酿酒酵母摄取铜机制的信息。观察到酵母细胞从生长培养基中摄取铜的两阶段动力学机制。第一阶段的摄取速率取决于生长培养基的初始铜浓度，持续约6小时。第二阶段的铜摄取速率比第一阶段慢，但动力学与初始铜浓度无关。直接从完整酵母细胞（在77K下）记录的发射光谱表明，铜在细胞内的掺入通过几种物种进行，最终导致铜以铜 - 金属硫蛋白的形式储存。在含铜培养基中生长的酵母细胞的显微照片清楚地显示出橙色发光，表明形成了硫醇铜（I）物种。通过从酵母细胞中排泄和分离含铜蛋白质后测量圆二色性和发射特性，证实了该物种为铜 - 金属硫蛋白。对77K下酿酒酵母铜 - 金属硫蛋白发射光谱的分析揭示了两个发射带，中心分别位于570和700nm。高能发射带表现出双组分衰减，激发态寿命分别为4.70和48.5微秒。低能发射表现出一个主要衰减组分，寿命为1.13微秒。还从酵母细胞中分离出一种高分子量的含铜物种，并对其进行了光谱表征。
