Poly(Adenosine Diphosphoribose) Polymerase in Mammalian Nuclei

The ability of isolated LS‐cell nuclei to incorporate [adenine‐3H]‐NAD into acid‐insoluble products is demonstrated. 2. The incorporation has a pH optimum at 8.5 and an apparent temperature optimum at 25 °C. 3. The optimum Mg2+ concentration is dependent upon the NAD concentration; at 1.5 μM NAD it is 2 mM; at 1.5 mM NAD it is 3 mM; and at 9 mM NAD it is above 20 mM Mg2+ Mg2+ can be replaced by Ca2+. 4. Dithiothreitol or mercaptoethanol, up to 10 mM, enhances the incorporation of NAD. 5. With the optimal conditions the incorporation of NAD (V) is 128 nmol NAD×5 min−1×mg DNA−1 and the Km is 1.47 ± 0.18 mM. Nicotinamide inhibits the incorporation competitively with a Ki of 14.3 ± 1 μM and thymidine inhibits competitively with a Ki of 32.5 μM. 6. The incorporation is not affected by added DNA or poly(U). It is not affected by ribonuclease but is inhibited by about 50% on treatment with deoxyribonuclease. 7. The enzyme system is unstable and the decay is temperature‐dependent; there is little decay at 0 °C but above this temperature the rate of decay follows an Arrhenius relationship. The initial rates of enzyme activity also show a temperature dependence with a linear Arrhenius plot. The temperature optimum at 25 °C is the arithmetic result of these two opposing processes.