Changes in the Activities of the Pentose Phosphate Pathway and Pyrimidine Nucleotide Biosynthesis during the Growth of Vinca rosea Cells in Suspension Culture

Abstract

Summary Four growth phases could be recognized from some growth parameters determined in a suspension cell culture of Vinca rosea which was subcultured every 10 days in the MURASHIGE-5KOOG medium. These are (I) lag phase (day 0-1), (II) phase of cell division (day 1–4), (III) cell expansion phase (day 4–7), and (IV) stationary phase (day 7–10). Prior to cell division, pronounced increase in respiration per cell occurred. The ratio of 14 CO 2 evolved from [6- 14 C]glucose to that from [1- 14 C]glucose (i. e., C 6 /C 1 ratio) decreased during the lag phase and reached a minimum during cell division phase and then increased again. Activities of the key enzymes of the pentose phosphate (PP) pathway, glucose- 6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) together with phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) increased coincidently just after inoculation and attained a maximum at the cell division phase, and the decreased. In contrast, the activity of a glycolytic enzyme, fructose-l,6-bisphosphate aldolase (EC 4.1.2.13) showed a different changing pattern during culture. RNA content per cell increased during lag phase and cell division phase and decreased during then two later growth phases, while no appreciable changes in DNA content occurred. Incorporation of [2- 14 C]uracil and [2- 14 C]uridine into the RNA fraction increased immediately after inoculation. The maximum rate of [2- 14 C]uracil incorporation was found at the end of the lag phase of cell growth and the rate decreased during the cell division phase. High levels of [2- 14 C]uridine incorporation remained during the cell division phase. Incorporation of [6- 14 C]orotate into the RNA fraction increased during the cell division phase and decreased during cell expansion and stationary phases. The pattern of change in the activity of carbamoylphosphate synthetase (EC 2.7.2.9) was similar to that in [6- 14 C] orotate incorporation into RNA. These results indicate that during the growth of Vinca cells in suspension culture, the PP pathway, PRPP synthesis, and uracil and uridine salvage pathways of pyrimidine nucleotide biosynthesis are activated during the lag phase and then the de novo pathway of pyrimidine nucleotide biosynthesis develops during the following cell division phase.