ACTION OF PARTIALLY THIOLATED POLYNUCLEOTIDES ON DNA POLYMERASE-ALPHA FROM REGENERATING RAT-LIVER

  • 1 January 1976
    • journal article
    • research article
    • Vol. 36  (12) , 4537-4542
Abstract
The effects of partially thiolated [antitumor] polynucleotides on the DNA polymerase .alpha. from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction according to the method of Baril et al. It was characterized as the .alpha. polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on DNA polymerase .alpha. as a template or an inhibitor, partially thiolated polycytidylic acid (MPC) was a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). DNA polymerase .beta., which was purified from normal rat liver nuclear fraction, was less sensitive to inhibition by MPC. Analysis of the inhibition of the .alpha. polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of 7.2% thiolated MPC to the enzyme was stronger than that of the template (Ki/Km < 0.03). Polyuridylic acid showed some inhibitory activity which increased on partial thiolation, but 8.4% thiolated polyuridylic acid was less active than 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double stranded configuration. One to 2%-thiolated DNA isolates were significantly more potent inhibitors than comparable (1.2% thiolated) MPC and showed competitive reversibility with the unmodified (but activated) DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition and other specific structural properties.

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