ABSTRACT
The effect of aflatoxin B1 on the DNA template and DNA-dependent RNA polymerases in buffalo liver was studied. Aflatoxin B1 inhibited both Mg2+- and Mn2+-activated RNA polymerases in a dose-dependent manner. At 10 micrograms the inhibition of both enzymes was almost complete. The inhibitory effect on the solubilized enzymes was higher than the chromatin-bound, suggesting a direct effect at the enzyme level. On the other hand, incubating DNA or deoxyribonucleoprotein (DNP) with 2 micrograms aflatoxin reduces its transcriptional capacity with a greater effect on the Mg2+-activated RNA polymerase than the Mn2+-activated enzyme. These results suggest that aflatoxin B1 inhibits in vitro transcription in buffalo liver at both enzyme and template levels.
Subject(s)
Aflatoxins/toxicity , Chromatin/enzymology , DNA-Directed RNA Polymerases/antagonists & inhibitors , DNA/drug effects , Liver/drug effects , Transcription, Genetic/drug effects , Aflatoxin B1 , Animals , Buffaloes , Female , In Vitro Techniques , Liver/metabolismABSTRACT
The cell-free extract prepared from Aspergillus flavus ATCC 5517/A 228 showed activity in converting sterigmatocystin to aflatoxin B1. The extract was purified on Ultrogel AcA-54 and resulted in ten protein peaks, one of which (peak VI) showed activity in sterigmatocystin conversion. The protein in this peak gave one protein band using polyacrylamide gel (PAG)-disc electrophoresis. For further purification, protein(s) in peak VI were applied on DEAE-Sephadex A-50 and two protein peaks were detected. Only one peak showed enzyme activity which showed homogeneity as one band on PAGE and sodium dodecyl sulphate (SDS)-PAGE. The optimum temperature for the enzyme activity was 28 degrees C and the optimum pH was 8. The maximum conversion resulted from the action of 0.6 mg enzyme protein on 48 X 10(-8) mol sterigmatocystin. Zn2+, Co2+ and Mn2+ enhanced the enzyme activity, while ethylenediaminetetraacetic acid, parahydroxymercuric benzoate and phenylmethylsulphonic fluoride inhibited the enzyme activity in a dose-dependent manner. Amino-acid analysis showed the presence of 22 amino acids, three of which are unknown. The enzyme has a molecular weight of 64,000 daltons (by gel filtration) and 70,000 daltons (by SDS-PAGE).
