The effect of copper and gallium compounds on ribonucleotide reductase
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.
- Research Organization:
- Medical Coll. of Wisconsin, Milwaukee, WI (United States)
- OSTI ID:
- 5581031
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
560300 -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
BIOCHEMISTRY
BIOLOGICAL EFFECTS
CARBOXYLIC ACID SALTS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHEMISTRY
CITRATES
COMPLEXES
COPPER COMPLEXES
CYTOCHEMISTRY
ELECTRON SPIN RESONANCE
ENZYME ACTIVITY
ENZYMES
GALLIUM COMPOUNDS
GALLIUM NITRATES
MAGNETIC RESONANCE
MEMBRANES
NITRATES
NITROGEN COMPOUNDS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDATION
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PROTEINS
RESONANCE
RNA
THIOLS
TRANSITION ELEMENT COMPLEXES