| Abstract: The mode of action of copper complexes (CuL and CuKTS) and gallium compounds (gallium nitrate and citrate) in cytotoxicity was studied.^The effects of these agents on the enzyme ribonucleotide reductase was investigated by monitoring the tyrosyl free radical present in the active site of the enzyme through electron spin resonance (ESR) spectroscopy.^Ribonucleotide reductase, a key enzyme in cellular proliferation, consists of two subunits.^M1, a dimer of molecular weight 170,000 contains the substrate and effector binding sites.^M2, a dimer of molecular weight 88,000, contains non-heme iron and tyrosyl free radical essential for the activity of the enzyme.^In studies using copper complexes, the cellular oxidative chemistry was examined by ESR studies on adduct formation with membranes, and oxidation of thiols.^Membrane thiols were oxidized through the reduction of the ESR signal of the thiol adduct and the analysis of sulfhydryl content.^Using the radiolabel [sup 59]Fe, the inhibitory action of copper thiosemicarbazones on cellular iron uptake was shown.^The inhibitory action of CuL on ribonucleotide reductase was shown by the quenching of the tyrosyl free radical on the M2 subunit.^The hypothesis that gallium directly interacts with the M2 subunit of the enzyme and displaces the iron from it was proven.^The tyrosyl free radical signal from cell lysates was inhibited by the direct addition of gallium compounds.^Gallium content in the cells was measured by a fluorimetric method, to ensure the presence of sufficient amounts of gallium to compete with the iron in the M2 subunit.^The enzyme activity, measured by the conversion of [sup 14]C-CDP to the labeled deoxy CDP, was inhibited by the addition of gallium nitrate in a cell free assay system.^The immunoprecipitation studies of the [sup 59]Fe labeled M2 protein using the monoclonal antibody directed against this subunit suggested that gallium releases iron from the M2 subunit. |
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