SYNTHESIS AND DEGRADATION OF NAD IN GUINEA-PIG CARDIAC-MUSCLE .2. STUDIES ABOUT THE DIFFERENT BIOSYNTHETIC PATHWAYS AND THE CORRESPONDING INTERMEDIATES

In continuation of the previous report the possible pathways for the incorporation of the precursors nicotinamide or nicotinic acid into NAD of isolated guinea pig atria have been analyzed by combined methods of paper- and column chromatography. After incubation with a high concentration of 20 mM14C-nicotinamide a deamidation was proved by finding high activities of14C-nicotinic acid in cardiac tissue. An important intermediate,14C-NaAD, could be also detected in a ratio of 1.35:1 related to the measured14C-NAD. Therefore for the new NAD synthesis the Preiss-Handler pathway is operating. No significant activity was found in NMN and NADP. From a total increase of 24.4 nMol NAD/8 h · 100 mg atrial tissue (+74%) 7.1 nMol can be attributed to a new synthesis of14C-NAD and 17.3 nMol NAD to the inhibition of the degradation. It is assumed that nicotinamide inhibits the NAD degrading glycohydrolase in high concentrations. After incubation with 20 mM14C-nicotinic acid only a very low14C-incorporation into NAD was observed with the Preiss-Handler pathway. The total NAD level does not change during an incubation time of 24 h. With a concentration of 10 μM14C-nicotinamide no deamidation to14C-nicotinic acid could be measured in atrial tissue. However, significant activities as14C-NMN,14C-NAD and14C-NADP were observed. Therefore the Dietrich pathway is operating here. With 10 μM14C-nicotinic acid a higher incorporation rate into NAD could be measured than was observed with 10 μM14C-nicotinamide during the same incubation period. The high radioactivity found in NaAD demonstrated that the synthesis proceeds here via the Preiss-Handler cycle. Under these conditions, however,14C-NAD can be degraded easily by the NAD glycohydrolase to14C-nicotinamide and ADPR.14C-nicotinamide was found in atrial tissue as well as in the nutrient solution and can be reincorporated into NAD via NMN in the Dietrich pathway, since significant radioactivity was observed in this intermediate. The analysis of the investigated precursors and intermediates of NAD synthesis and degradation could be established only by the combined methods of paper- and column chromatography, because14C-NaAD can be separated from14C-NAD only with column chromatography and14C-nicotinic acid from14C-NAD only with paper chromatography. © 1979 Dr. Dietrich Steinkopff Verlag.