Ribonucleotide reductase in dividing cells : purification and inhibition studies with 4-hydroxynonenal
1). The effect of temperature, P450 inhibitors (pyrazole and imidazole), sulphydryl reagents (iodoacetamide and N-ethyl maleimide) and glutathione on the activation of CCl4 in rat liver microsomes was studied. Spin trapping of CCI3', covalent binding of CCl4 to protein and CCl4-dependent MDA formation were used as indices of CCl4 metabolism. Formation of PBN-CCI3' adduct, 14CCl4 covalent binding to protein and CCl4-dependent :MDA production were dependent on temperature range from 15-40°C. The transition temperature was at 26.7 -27 .5°C when the activation was measured by formation of PBNCCl3' adduct and specific 14CCl4 covalent binding. The transition temperature was found to be 34.3°C when CCl4 -dependent MDA production was taken as the index of the activation of CCI4. Pyrazole, imidazole and iodoacetamide inhibited CC14 -dependent MDA formation only at high concentrations (10-20 mM), whereas glutathione showed a strong inhibitory effect on CCl4-stimulated lipid peroxidation. MDA formation was nearly 100°;6 inhibited by 1 roM GSH. GSH also delayed the onset of lipid peroxidation. N-ethyl maleimide (NEM) exerted biphasic effects on CCl4 -dependent MDA formation. The lower concentration of NEM (0.5 mM-l mM) reduced the :MDA prodUction, while the higher concentration of NEM (5-10 mM) enhanced the MDA formation. 2). Ribonucleotide reductase was partially purified from juvenile normal rat liver. The enzyme was purified 30 fold after DEAE-cellulose chromatography. The CDP reductase activity in tissues with different growth states or rates was compared. The enzyme activity was developed well in juvenile rat liver, regenerating liver and hepatoma (cells), while the enzyme activity was undetectable in adult rat liver and sham-operated rat liver. The enzyme activity in Yoshida cells was 3-fold of the activity in Morris 5123tc tumours. Dithiothreitol (DIT) activated the activity of CDP reductase from 48h and 60h regenerating liver, but DIT did not activate the enzyme activity of juvenile 'normal rat liver. The possible mechanism of the activation of enzyme activity by DIT was discussed and a mechanism of regulation of the ribonucleotide reductase activity in regenerating liver was suggested. 3). The effect of the lipid peroxidation product 4-hydroxynonenal (HNE) on CDP reductase from juvenile normal rat liver was investigated. HNE inhibited the CDP reductase activity. The inhibition was dependent on the concentration of HNE and the incubation time. The enzyme activity was reduced 500/0 by 0.1 roM HNE. The inhibitory effect of HNE was irreversible. DIT protected the enzyme against HNE suggesting that HNE inhibited the activity of ribonucleotide reductase from rat liver through the mechanism of blockage of functional SH groups in the enzyme protein.