| Popis: |
In the blue crab Callinectes sapidus , a direct correlation exists between the levels of Cu(I)-metallothionein (CuMT) and Cu(I)-hemocyanin during the molt cycle. However, CuMT cannot directly restore the oxygen-binding capacity of copperless hemocyanin. Here we report the discovery of an intracellular ligand that may facilitate the transfer of Cu(I) from CuMT to the active site of apohemocyanin. The tripeptide glutathione (γ-Glu-Cys-Gly, GSH) in the digestive gland of the American lobster, Homarus americanus , co-elutes with copper-metallothionein during size-exclusion chromatography. The latter protein can be separated into three isoforms by anion-exchange chromatography: CuMT-I, CuMT-II, and CuMT-III, as has been found for the CuMTs from the blue crab. The interaction between GSH and MT isoforms was examined by ultrafiltration experiments and size-exclusion HPLC. CuMT-III forms a stable 1:1 complex with GSH, with a dissociation constant of 1 μm. CuMT-I and II make a transient complex with GSH, with subsequent release of copper as a copper-glutathione [Cu(I)-(GSH) 2 ] complex. This complex can restore oxygen binding by apohemocyanin. GSH also forms a complex with cadmium-zinc MT from rabbit liver and can repair the oxidized form of this protein. The binding site for GSH on the mammalian MT has been tentatively identified by molecular-graphics analysis and energy-minimization calculations. These studies suggest that MT and GSH are intricately linked in the biochemistry of metal regulation. |
