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  • Title: Regulation of nucleotide and pentose synthesis in resting and stimulated 3T6 fibroblasts.
    Author: Buchanan JM, Smith ML, Smith RJ.
    Journal: Adv Enzyme Regul; 1982; 20():135-52. PubMed ID: 6180605.
    Abstract:
    A two-step procedure has been used to follow the activation of one metabolic system involved in the return of cells to a proliferative state after resting in a Go state as a result of serum limitation. One feature of the resting state is a limited capacity to synthesize nucleotides. The limitation apparently is in the rate of synthesis of 5-phosphoribosylpyrophosphate from glucose and indirectly in the capacity of the resting cells to turn over the triphosphopyridine nucleotide pair, NADPH:NADP+. A reaction utilizing NADPH is apparently greatly diminished in resting cells and is substantially increased by only brief contact of cells with the hormonal elements in dialyzed calf serum. Insulin together with platelet-derived growth factor can substitute for calf serum. Aside from stimulating the turnover of the pyridine nucleotide coenzyme pair, serum also stimulates the utilization and reformation of ATP, principally from AMP. Among the NADPH-linked reactions that have been examined for their physiological significance in the initiation of growth stimulation are two steps in the conversion of glutamate to proline in the cytoplasm. Pyrroline 5-carboxylate, an intermediate in this metabolic pathway, has been shown to stimulate PRPP synthesis when added to cultures of resting 3T6 cells. Proline, the product of the reduction of this 5-membered heterocycle is also a stimulant of PRPP synthesis. In addition, dehydroascorbic acid is a potent stimulant of PRPP synthesis. As a working hypothesis, we are exploring the role of a series of reactions that form a pyrroline 5-carboxylate/proline cycle operating between the cytoplasm and mitochondria. The net result is the oxidation of NADPH by molecular oxygen to yield NADP+ and water. The NADP+ is then used in the hexose monophosphate pathway for the conversion of glucose to PRPP. We wish to determine whether dehydroascorbate is operating in this cycle as an oxidant of proline in the mitochondria or whether it participates in some other reaction in the cell that redistributes the ratio of NADPH to NADP.
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