The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP.

Zn(II) complexes of 1-oxa-4,7,10-triazacyclododecane (12[ane]N3O), 1,5,9-triazacyclododecane (12[ane]N3), and 1-hydroxyethyl-1,4,7-triazacyclononane (9[ane]N3OH) promote cleavage of the RNA analogue, 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) at pH 8.0, I=0.10 M (NaCl), 25 degrees C with second-order rate constants of 8.9x10(-3), 9.0x10(-3), and 3.3x10(-3) M-1 s-1, respectively. Cleavage of HpPNP by these catalysts is inhibited by uridine with inhibition constants (Ki) of 1.2, 0.46, and 45 mM, respectively, under these conditions. Binding constants derived from these inhibition constants are 2-200-fold larger than those for binding of related Zn(II) complexes to phosphate diesters under similar conditions, suggesting that uridine sequences in RNA will inhibit Zn(II)-catalyzed cleavage by competing with phosphate diester binding sites. Further studies are carried out that utilize pH-potentiometric titrations to monitor uridine binding to five Zn(II) macrocyclic complexes in aqueous solution at 25 degrees C, I=0.10 M (NaCl). The data are consistent with binding of the Zn(II) complexes to the N3-deprotonated form of uridine to give log KU.-values of 5.29, 4.57, 4.56, 3.47, and 2.65 for the Zn(II) complexes of 12[ane]N3, 12[ane]N4, 12[ane]N3O, 15[ane]N3O2, and 9[ane]N3OH, respectively (12[ane]N4=1,4,7,10-tetraazacyclododecane, 15[ane]N3O2=1,4-dioxa-7,10,13-triazacyclopentadecane). For the five Zn(II) complexes studied, there is a linear relationship between uridine anion binding constants and hydroxide binding constants.

Mononuclear complexes between Zn(2+) and the following four macrocycles were prepared: 1,4,7,10-tetraazacyclododecane (1), 1-oxa-4,7,10-triazacyclododecane (2), 1,5,9-triazacyclododecane (3) and 1-hydroxyethyl-1,4,7-triazacyclononane (4). The pH rate profiles of values of the observed second-order rate constant log (k(Zn))(app) for Zn(X)(OH(2))-catalyzed cleavage (X = 1, 2, 3 and 4) of 2-hydroxypropyl-4-nitrophenyl phosphate (HpPNP) show downward breaks centered at the pK(a) for ionization of the respective zinc bound water. At low pH, where the rate acceleration for the catalyzed reaction is largest, the stabilizing interaction between the catalyst and the bound transition state is 5.7, 7.4, 7.4 and 5.9 kcal mol(-1) for the reactions catalyzed by Zn(1)(OH(2)), Zn(2)(OH(2)), Zn(3)(OH(2)) and Zn(4)(OH(2)), respectively. The interactions between the metal cation and the macrocycle cause either a modest increase or reduction in transition state stabilization compared with 6.6 kcal mol(-1) stabilization for catalysis by Zn(OH(2))(6). The best Zn(II)-macrocycle catalysts are those for which the interactions between the metal ion and macrocycle are the weakest. Inhibition studies show that each of the four catalysts form complexes with phosphate and oxalate dianions with a much higher affinity than diethyl phosphate monoanion, consistent with stronger interaction of the catalysts with the transition state dianion compared with the substrate monoanion HpPNP. The pH-dependence of methyl phosphate inhibition of Zn(2) catalyzed cleavage of HpPNP shows that only the Zn(2)(OH(2)) species binds the inhibitor. This result is consistent with a mechanism that has Zn(2)(OH(2)) as the active catalytic species.

A new spectroscopic system for direct photoluminescence of lanthanide ions (Ln(III)) through electronic transitions within the 4f(n) manifold is described. The system is based on an injection seeded frequency tripled (lambda = 355 nm) Nd:YAG pump laser coupled with a master oscillator power oscillator (MOPO). The MOPO delivers an average pulse energy of approximately 60 mJ/pulse, is continuously tunable from 425 to 690 nm (Signal) and 735 to 1800 nm (Idler) with a linewidth of <0.2 cm(-1), and has a pulse duration of 10-12 ns. Aqueous solutions containing two polyaminocarboxylate complexes, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), and Ln(3+) aqua ion for several lanthanides including Eu(III), Tb(III), Dy(III), and Sm(III)) are used as steady-state and time-resolved photoluminescence standards. The versatility of the instrument is demonstrated by excitation scans over a broad visible range for aqueous solutions of complexes of Eu(III), Dy(III), Sm(III), and Tb(III). The Eu(III) excitation band ((7)F(o)-->(5)D(o)) is recorded over a range of complex concentrations that are 1000-fold less than reported previously, including Eu(EDTA) (1.00 nM), Eu(DTPA) (1.00 nM), and Eu(III) aqua ion (50.0 nM). Emission spectra are recorded in the visible range for Ln(III) complexes at pH 6.5 and 1.00 mM. Excited-state lifetimes for the standards were constant as a function of concentration from 10.0 nM to 1.00 mM for Eu(EDTA) and Eu(DTPA) and from 100 nM to 1.00 mM for Eu(III) aqua ion. Photoluminescence lifetimes in H(2)O and D(2)O are recorded and used to calculate the number of bound water molecules for all complexes.

The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.