Probing the coordination environment of the human copper chaperone HAH1: characterization of Hg(II)-bridged homodimeric species in solution.

Although metal ion homeostasis in cells is often mediated through metallochaperones, there are opportunities for toxic metals to be sequestered through the existing transport apparatus. Proper trafficking of Cu(I) in human cells is partially achieved through complexation by HAH1, the human metallochaperone responsible for copper delivery to the Wilson and Menkes ATPase located in the trans-Golgi apparatus. In addition to binding copper, HAH1 strongly complexes Hg(II), with the X-ray structure of this complex previously described. It is important to clarify the solution behavior of these systems and, therefore, the binding of Hg(II) to HAH1 was probed over the pH range 7.5 to 9.4 using (199)Hg NMR, (199m)Hg PAC and UV-visible spectroscopies. The metal-dependent protein association over this pH range was examined using analytical gel-filtration. It can be concluded that at pH 7.5, Hg(II) is bound to a monomeric HAH1 as a two coordinate, linear complex (HgS2), like the Hg(II)-Atx1 X-ray structure (PDB ID: 1CC8). At pH 9.4, Hg(II) promotes HAH1 association, leading to formation of HgS3 and HgS4 complexes, which are in exchange on the µs-ns time scale. Thus, structures that may represent central intermediates in the process of metal ion transfer, as well as their exchange kinetics have been characterized.

Contribution of whole blood to the control of plasma asymmetrical dimethylarginine.

The endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is elevated in many patients and may contribute to the initiation and progression of their disease. While some mechanistic pathways have been identified, tissue-specific contributions to ADMA control remain unclear. We sought to determine if whole blood (WB) could participate in ADMA control ex vivo. Anesthetized male Sprague-Dawley rats underwent exsanguinations, and WB preparations were incubated at 37 degrees C for 5 h. ADMA and symmetrical dimethylarginine were analyzed by high-pressure liquid chromatography. Incubation of lysed red blood cell (RBC) supernatant yielded a significant decrease in ADMA that was blocked by 4124W, a synthetic inhibitor of dimethylarginine dimethylaminohydrolase, the only reported enzyme to hydrolyze ADMA. Hydrolysis of ADMA was diminished by addition of physiologically relevant concentrations of zinc (i.e., 20 microM). Conversely, when rat WB or WB supernatant was incubated at 37 degrees C, it liberated quantities of free ADMA (1-2 microM) that in vivo would likely have pathological consequences. Addition of arginine methyltransferase inhibitors to these incubations did not reduce ADMA release, indicating no dominant role for active protein methylation during these incubations. This ADMA liberation was significantly reduced by addition of protease inhibitors, indicating a dependence on peptide bond hydrolysis. Total ADMA (protein incorporated plus free) was determined by acid hydrolysis and found to be 43.18 +/- 4.79 microM in WB with approximately 95% of this in RBCs. These ex vivo data demonstrate the potential of blood to control the NO-NOS system by modulating free ADMA.