ISOLATION, PURIFICATION, AND CHARACTERIZATION OF ZINC-INDUCED, METAL-BINDING PROTEIN FROM LIVER OF RAINBOW TROUT (SALMO GAIRDNERI).

摘要

Zinc is both a ubiquitous, aquatic pollutant and an element essential to maintain the health of fishes. This dual role of zinc exemplifies the need to understand the biochemical and physiological mechanisms involved in metal metabolism by fishes. The inducible protein, metallothionein, binds up to seven zinc atoms per molecule and potentially could serve as a key regulatory step between zinc metabolism and toxicity. The purpose of this research was to isolate, purify, and characterize metallothionein from salmonid fishes, a taxon noted for its acute sensitivity to zinc and other heavy metals.; Metal-binding protein was induced in the liver and kidney of rainbow trout, Salmo gairdneri, using 7 mg/kg intraperitoneal injections of zinc(II). Liver metal-binding protein was isolated using Sephadex chromatography; the purity of the metal-binding protein was verified by sodium dodecyl sulfate electrophoresis. Synthesis of liver metal-binding protein peaked at 24 hr following one zinc injection and increased linearly with temperature between 9 and 17(DEGREES)C, but was inhibited by cycloheximide. The purified liver metal-binding protein has a molecular weight of 12,400 daltons (gel filtration), and its amino acid composition included 7% cysteine, several aromatic amino acids, and ornithine. If one assumes that the metal-binding protein had a molecular weight of 6,621 daltons, then each mole of the metal-binding protein bound four moles of zinc and contained 18 moles of sulf-hydryl (-SH) groups. The liver metal-binding protein was tentatively identified as a non-thionein metal-binding protein. The absence of metallothionein from rainbow trout liver may partially explain the sensitivity of rainbow trout to heavy metal divalent ions.; A second metal-binding protein was also isolated from the liver preparations using the techniques described above. This protein probably was a dimer with a molecular weight of 32,000 daltons which consisted of two identical subunits. Its amino acid composition suggested that the protein may be superoxide dismutase, an enzyme that converts superoxide radicals into hydrogen peroxide. Low concentrations of this enzyme probably cause the faint secondary protein band on electrophoretic gels noted by many workers purifying metallothionein from fish liver.