RESUMO
Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on the avian myeloblastosis virus reverse transcriptase activity. The drug inhibited the DNA synthesis reaction catalyzed by the viral enzyme in the presence of different template-primers. The inhibitory effect by nalidixic acid was higher with polyriboadenylic acid than with polyribocytidylic acid as a synthetic template. With activated DNA as a template nalidixic acid preferentially inhibited the TMP incorporation when compared with the dAMP incorporation. Both these results showed the importance of the presence of adenine in the templates for a more efficient inhibition by nalidixic acid. The inhibition for this drug was also shown in the presence of Mn2+ instead of Mg2+ as the divalent cation, and with a 2'-fluorinated analogue of polyriboadenylic acid as the template. Kinetic data showed a non-competitive inhibition by nalidixic acid in relation to polyriboadenylic acid and to TTP in the reaction catalyzed by reverse transcriptase.
Assuntos
Vírus da Mieloblastose Aviária/efeitos dos fármacos , Ácido Nalidíxico/farmacologia , Inibidores da Transcriptase Reversa , Vírus da Mieloblastose Aviária/enzimologia , Vírus da Mieloblastose Aviária/genética , DNA Viral/biossíntese , Cinética , Polinucleotídeos/síntese química , Polinucleotídeos/metabolismo , Moldes GenéticosRESUMO
Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different Km, Vmax and were strikingly insensitive to 5-fluorouracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10-100-fold higher concentrations of each analogue to achieve comparable inhibition of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.