Role of nucleosides, 5-phosphoribosyl-1-pyrophosphate, and ribose 1-phosphate in the biosynthesis of phosphosphingolipids in Bacteroides melaninogenicus.

Using a deenergized spheroplast system from Bacterioides melaninogenicus, the sphingolipid precursor 3-ketodihydrosphingosine is not incorporated into the complete sphingolipids, ceramide phosphorylethanolamine, or ceramide phosphorylglycerol unless supplied with glutamine, ATP, ADP, or AMP. Adenosine, inosine, and certain other nucleosides were as effective as ATP. Purine bases and ribose, however, were inactive in this system. 5-Phosphoribosyl-1-pyrophosphate and ribose 1-phosphate also stimulated conversion of 3-ketodihydrosphingosine into ceramide phosphorylethanolamine and ceramide phosphorylglycerol, whereas ribose 5-phosphate showed only slight activity. Hypoxanthine was the main product formed from inosine and adenosine but there was no evidence for nucleotide formation. Adenosine stimulated³²P_i incorporation into cell phospholipids indicating that ribose 1-phosphate, formed via purine nucleoside phosphorylase, could be the compound stimulating sphingolipid synthesis in this system.     

Stimulation of ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate generation by pyrroline-5-carboxylate in mouse liver in vivo: evidence for a regulatory role of ribose-5-phosphate availability in nucleotide synthesis.

Pyrroline-5-carboxylase (P5C), a physiological stimulator of hexose-monophosphate-pentose pathway activity, was found before to increase 5-phosphoribosyl-1-pyrophosphate (PRPP) generation and nucleotide synthesis in human erythrocytes and cultured fibroblasts. We now report the stimulation of PRPP generation by P5C also in mouse liver in vivo. In addition we demonstrated a simultaneous elevation in ribose-5-phosphate (R5P) concentration, which was relatively smaller and transient. The demonstrated effect of P5C on liver R5P and PRPP content in vivo provides strong evidence for the physiological role of R5P availability in the regulation of PRPP and purine production.     

Modulation of glycogen phosphorylase activity affects 5-phosphoribosyl-1-pyrophosphate availability in rat hepatocyte cultures

The effect of modulation of the rate of glycogenolysis on the availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) was investigated in rat hepatocyte cultures. Dibutyryl cyclic AMP (dbcAMP), forskolin and glucagon, activating glycogen phosphorylase through activation of protein kinase A (PKA), were found to raise PRPP availability by 44%-56%. Arg-vasopressin and phenylephrine, activating glycogen phosphorylase through the phosphoinositide cascade, did not affect PRPP availability. dbcAMP, but not phenylephrine, increased the degradation of pre labeled glycogen by 57%. Caffeine and CP-91149, inhibitors of glycogen phosphorylase, decreased PRPP availability by 33% and 43%, respectively. The finding that induction of glycogenolysis enhances, and inhibition of glycogenolysis decelerates PRPP generation suggests that glycogenolysis is a major contributor to PRPP generation in liver tissue in the basal (postabsorptive) state.     

Effects of glycerol and fructose on purine synthesis de novo and on PP-ribose-P availability in rat liver cells

Incubation of freshly isolated rat liver cells with glycerol resulted in an initial decrease, followed by an increase in purine synthesis de novo and in PP-ribose-P availability. The magnitude of these effects was dependent on the concentration of glycerol; as it increased, the initial period of latency or inhibition was prolonged, and the extent of the subsequent stimulation was greater. The intracellular Pi concentration and the [14C]ATP/[14C]ADP ratio were also initially decreased in these cells, and they too returned subsequently to normal values. All these changes were similar to those induced by fructose under the same conditions. The increase in PP-ribose-P availability always preceded that in purine synthesis de novo, indicating that, under most circumstances, PP-ribose-P availability is limiting for purine synthesis de novo. Finally, PP-ribose-P synthesis in these cells varied in parallel with the intracellular Pi concentration and with the ATP/ADP and ATP/AMP ratios.     

Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate

Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.     

Effect of inorganic phosphate on synthesis of 5-phosphoribosyl-1-pyrophosphate in cultured plant cells

• 1.1. Inorganic phosphate (Pi) was absorbed rapidly by suspension-cultured cells of Catharanthus roseus which had previously been cultured in Pi-free Murashige Skoog medium.
• 2.2. The intracellular levels of ATP, ADP and 5-phosphoribosyl-l-pyrophosphate (PRPP) increased markedly during the 24 hr which followed the addition of Pi (1.25mM).
• 3.3. Availability of PRPP in vivo, estimated by the measurement of nucleotide synthesis from [8-¹⁴C]adenine, was also increased by addition of Pi.
• 4.4. Only a 20% increase in the maximum catalytic activity of PRPP synthetase was observed in extracts of cells, prepared 24 hr after addition of Pi.
• 5.5. In contrast to results for mammalian PRPP synthetase, the activity of PRPP synthetase, partially purified from Catharanthus roseus, was inhibited by concentration of Pi greater than 5mM.
• 6.6. The mechanisms involved in the increased availability of PRPP and the synthesis of adenine nucleotides in the plant cells cultured in Pi-containing medium are discussed.

     

Rat liver glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase [EC 2.4.2.14]. Purification and properties.

Glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase (amidophosphoribosyltransferase), [EC 2.4.2.14] was purified 1,600-fold from rat liver. The preparation gave two protein bands on acrylamide gel electrophoresis, of which only the main band showed enzyme activity. The molecular weight of the enzyme was estimated to be 215,000, 200,000, and 195,000 by Sephadex G-150 gel filtration, polyacrylamide gel electrophoresis, and sucrose density grandient ultracentrifugation, respectively. The apparent Km values for glutamine and PRPP were 1.24 mM and 0.57 mM, respectively. The concentration-activity curve for PRPP changed from a hyperbolic to a sigmoidal form on addition of AMP or GMP, and this inhibition by AMP was prevented by increasing the PRPP concentration. In the presence of high concentrations of inorganic phosphate, the catalytic activity was decreased and the sensitivity to AMP inhibition was slightly increased. The molecular size of liver amidotransferase was not changed by the addition of PRPP, AMP, or 2-mercaptoethanol. The purified rat liver enzyme has a broad pH-range of activity between 6.5 and 8.5.     

Purine and pyrimidine salvage in whole rat brain. Utilization of ATP-derived ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate generated in experiments with dialyzed cell-free extracts.

The object of this work stems from our previous studies on the mechanisms responsible of ribose-1-phosphate- and 5-phosphoribosyl-1-pyrophosphate-mediated nucleobase salvage and 5-fluorouracil activation in rat brain (Mascia, L., Cappiello M., Cherri, S., and Ipata, P. L. (2000) Biochim. Biophys. Acta 1474, 70-74; Mascia, L., Cotrufo, T., Cappiello, M., and Ipata, P. L. (1999) Biochim. Biophys. Acta 1472, 93-98). Here we show that when ATP at "physiological concentration" is added to dialyzed extracts of rat brain in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uracil-, and 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nucleotide synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP even in the absence of added pentose or pentose phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through the action of purine nucleoside phosphorylase, phosphopentomutase, and 5-phosphoribosyl-1-pyrophosphate synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1-pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L., and Ipata, P. L. (2001) Biochem. Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major role in modulating adenine salvage in rat brain. On the basis of our in vitro results, we suggest that massive ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5-phosphoribosyl-1-pyrophosphate and ribose-1-phosphate pools, to be utilized for nucleotide resynthesis during reperfusion.     

Regulation of 5-phosphoribosyl-1-pyrophosphate synthesis in human fibroblasts by the concentration of inorganic phosphate

During the growth cycle of normal fibroblasts and of fibroblasts deficient in glucose-6-phosphate dehydrogenase activity, the concentration of 5-phosphoribosyl-1-pyrophosphate and of P_i, as well as the activity of 5-phosphoribosyl-1-pyrophosphate synthetase, decreased to stable values in confluent cultures. A high degree of correlation (0.89 and 0.91 for two normal and 0.69 for one glucose-6-phosphate dehydrogenase-deficient cell strain, respectively) was shown between intracellular P_i and 5-phosphoribosyl-1-pyrophosphate concentrations under varying culture and incubation conditions. 5-phosphoribosyl-1-pyrophosphate concentrations were elevated in normal fibroblasts incubated with methylene blue only if intracellular P_i levels were high. Neither methylene blue nor 6-aminonicotinamide, singly, affected intracellular P_i concentrations. However, when normal cells were pretreated with 6-aminonicotinamide and then with methylene blue, intracellular P_i decreased, 5-phosphoribosyl-1-pyrophosphate was depleted, and its rate of generation decreased. Under similar conditions, glucose-6-phosphate dehydrogenase-deficient fibroblasts maintained unaltered P_i levels, and 5-phosphoribosyl-1-pyrophosphate concentration and generation were slightly increased. The decrease in intracellular P_i in normal cells after the combined treatment was commensurate with an accumulation of 6-phosphogluconate, which did not take place in mutant cells. The changes in 5-phosphoribosyl-1-pyrophosphate synthesis, whether due to the stage of growth or various experimental manipulations, were always concordant with changes in intracellular P_i level. The regulatory role of P_i is consistent with the known enzymic properties of 5-phosphoribosyl-1-pyrophosphate synthetase.     

The effects of adenine nucleotides and guanine nucleotides on urate synthesis de novo by isolated chick liver and kidney cells.

Isolated chick liver and kidney cells produce urate de novo from glycine, and this is partially inhibited by 1 mm-AMP and by 1 mm-GMP in liver cells but not in kidney cells. Azaserine fully inhibits this synthesis de novo, but attempts to isolate formylglycine amide ribonucleotide from azaserine-blocked cells were unsuccessful.     

Electrophoretic heterogeneity of 5-phosphoribosyl-1-pyrophosphate synthetase within and among humans

The product of the enzyme 5-phosphoribosyl-1-pyrophosphate (PPriboseP) synthetase is a substrate for purine, pyrimidine, and pyridine biosynthesis and may be rate limiting for purine biosynthesis. A system developed to electrophoretically separate and histochemically detect PPriboseP synthetase in crude cell extracts has facilitated the identification of electrophoretically variant enzyme forms in the erythrocytes of five patients from a patient population of 200. Additional studies of human organs obtained at autopsy revealed a unique electrophoretic mobility for nearly each organ within the same individual.     

Effect of testosterone on purine synthesis de novo in rat perineal muscle in vivo

We examined in vivo the influence of testosterone on purine synthetis de nov, in the levator ani and gastrocnemius muscles of the rat. The hypoxanthine, adenine and guanine contents and the rate of incorporation of [¹⁴C]formate into these purine bases were determined in castrated adult and prepubertal rats (groups 1 and 2) both before and after orchiectomy and, in the second case, at different times after testosterone treatment. Substantially similar behavior was found in both groups, with some specific differences. The results showed an increase in the basal levels after castration (except for a dramatic decrease in adenine and a rise in the Gua/Ade molar ratio in prepubertal rats) and a return to basal levels after hormone administration, which was also accompanied by variations in the Gua/Ade molar ratio. The kinetics of purine nucleotide synthesis de novo and, spefically, of the overall reactions: IMP formation from PRib-PP, IMP → AMP and IMP → GMP, were followed by evaluating the incorporation curves of [¹⁴C]formate into hypoxanthine, adenine and guanine. Our results show that testosterone administration enhanced the incorporation rate and gave characteristic patterns: a diphasic cyclic oscillation of the Ade values in adult castrated rats, and single peaks having a specific shape in the other cases. The Gua/Ade labeling ratio was unchaned in castrated rats and increased in both groups during ther first 5 days after testosterone treatment, after which values even fell below normal; in most cases, values overlapped the pattern of the Gua/Ade molar ratio. The specific profile of the curves indicated that testosterone initially accelerated the turnover of guanylic acid and in the second phase re-established the normal behavior and ratio of AMP and GMP formation. These results indicate that the ‘inosinic branch point’ was subject to regulation by testosterone. The profiles of the incorporation curves and of the Gua/Ade ratio were indicative of a primary and secondary response to hormone action.