De novo and salvage biosynthetic pathways of pyridine nucleotides and nicotinic acid conjugates in cultured plant cells

To estimate the operation of the de novo and salvage pathways of pyridine nucleotide synthesis, [3H]quinolinic acid, an intermediate of the de novo synthesis, and [14C]nicotinamide and [14C]nicotinic acid, substrates of the salvage pathways, were administered to cultured white spruce (Picea glauca) and Madagascar periwinkle (Catharanthus roseus) cells and overall metabolism of these radioactive compounds was examined over the culture period. In P. glauca cells, all three precursors were able to generate pyridine nucleotides (mainly NAD and NADP) and trigonelline. Supplied [3H]quinolinic acid was efficiently converted to pyridine nucleotides and trigonelline in both at the logarithmic and the stationary stages of cell growth, although uptake of [3H]quinolinic acid by P. glauca cells was very low. [14C]Nicotinic acid and [14C]nicotinamide were taken up by the cells in a relatively facile manner, and 15–20 and 32–58% of total radioactivity from these compounds was found in pyridine nucleotides (mainly NAD and NADP) and trigonelline, respectively, after 18 h incubation. In C. roseus cells, these three precursors were utilised for pyridine nucleotides, but in contrast to P. glauca, nicotinic acid glucoside, but not trigonelline, was heavily labeled. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid. In Pi-starved cells, the uptake of quinolinic acid, nicotinic acid and nicotinamide was markedly decreased. Pyridine nucleotide synthesis de novo was greatly reduced in Pi-starved C. roseus cells, while little effect was found in the salvage pathway of nicotinic acid. Pi-deficiency slightly increased the rate of nicotinic acid-glucoside synthesis from nicotinic acid and nicotinamide. From the in vitro determination of enzyme activity, it is concluded that quinolinic acid and nicotinic acid are converted to nucleotides by quinolinate phosphoribosyltransferase (2.4.2.19) and nicotinate phosphoribosyltransferase (2.4.1.11), respectively. High activity of nicotinamidase (3.5.1.19) but no detectable activity of nicotinamide phosphoribosyltransferase (2.4.2.12) suggests that nicotinamide is converted to nicotinic acid, and then salvaged by nicotinate phosphoribosyltransferase.