| Autor: |
Liuchun Yang, McRae, Reagan, Henary, Maged M., Patel, Raxit, Lai, Barry, Vogt, Stefan, Fahrni, Christoph J. |
| Předmět: |
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| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; 8/9/2005, Vol. 102 Issue 32, p11179-11184, 6p |
| Abstrakt: |
Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of ΛG‡ = 44 kJ.mol-1 and kobs ∼ 105 s-1 at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensors copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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