A sensitive method for estimating 5-phosphoribosyl 1-pyrophosphate in Escherichia coli

A sensitive method, which uses a PRT'ase-catalysed reaction to couple PRPP with a labeled base, has been described for estimating the PRPP content of E. coli. Although the method is basically that of Henderson and Khoo (2), a new chromatographic system, which allows the complete separation of [¹⁴C]nucleoside 5′-phosphate from any contaminating [¹⁴C]-labeled base, has been devised. Further, the extraction process and the conditions for the PRT'ase reaction have both been modified for application to bacterial cultures. Finally, a choice between methods using either [¹⁴C]adenine or [¹⁴C]guanine, with their respective PRT'ase enzymes, allows for the estimation of PRPP in extracts which contain unlabeled purine or pyrimidine bases.     

The PRPP synthetase spectrum: what does it demonstrate about nucleotide syndromes?

Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme "superactivity" (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense mutations producing loss-of-function); (4) moderate PRS-I deficiency/Charcot-Marie-Tooth disease-5 (less severe loss-of-function mutations); and (5) mild PRS-I deficiency/Deafness-2 (mutations producing slight destabilization). Similar to Lesch-Nyhan disease, PRPS1-related disorders arise from phosphoribosyl-pyrophosphate (PRPP)-dependent nucleotide "depletion" of purine nucleotides (e.g., ATP, GTP). S-adenosylmethionine (SAMe) appears to partially alleviate purine depletion via a PRPP-independent path. Synthesis of pyrimidine nucleotides is PRPP dependent, with uridine monophosphate synthase deficiency producing pyrimidine nucleotide depletion. But pyrimidine salvage from uridine does not require PRPP, and this nucleoside is transported freely to pyrimidine-depleted tissues. Regulation of nicotinamide nucleotides is less clear; synthesis from pyridine nucleobases is PRPP dependent. Nucleotide "depletion" contrasts with nucleotide "toxicity," exemplified by the purine disorders adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies or by pyrimidine nucleotidase deficiency. These are characterized by the accumulation of one or more abnormal nucleotides such as succinyl- or deoxy-nucleotides or their metabolites, which interrupt other nucleotide or related pathways or are toxic to specific cell types. Theoretically, purine toxicity disorders would not be ameliorated by SAMe therapy, and this was confirmed for one adenylosuccinate lyase-deficient child. Nucleotide defects may also be seen as an aspect of mitochondrial disease, with SAMe-based mitochondrial therapy perhaps meriting further investigation.     

UMP pyrophosphorylase of Tetrahymena pyriformis. Partial purification and properties.

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K_m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 mm, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.     

The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 μM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP form Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.     

Determination of the intracellular concentration of 5-phosphoribosyl-1-pyrophosphate in cultured mammalian fibroblasts

The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of ¹⁴CO₂ from [carboxyl-¹⁴C]orotic acid by a commercially available preparation of yeast orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5′-nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained $\text{7.76 ± 1.14 nmol of PRPP}\text{10}{}^7\text{cells}$.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5′-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP:d-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5′-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di-, and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl₂ to promote PRPP synthetase subunit aggregation. Increasing the MgCl₂ to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

The control of synthesis of bacterial cell walls. Interaction in the synthesis of nucleotide precursors

Phosphoenolpyruvate-UDP-N-acetylglucosamine enolpyruvyltransferase, UDP-N-acetylglucosamine pyrophosphorylase and CDP-glycerol pyrophosphorylase activities were demonstrated in soluble extracts from Bacillus licheniformis A.T.C.C. 9945. The effect of various nucleotides, sugar nucleotides and sugar phosphates on the nucleotide pyrophosphorylases was investigated. UDP-N-acetylglucosamine pyrophosphorylase was inhibited by UDP-MurAc-pentapeptide (UDP-N-acetylmuramyl-l-alanyl-d-glutamyl- meso-diaminopimelyl-d-alanyl -d-alanine) and CDP-glycerol. CDP-glycerol pyrophosphorylase was inhibited by UDP-MurAc-pentapeptide and stimulated by UDP-N-acetylglucosamine. Interaction between a precursor of one cell-wall polymer and an enzyme involved in the synthesis of a precursor of a second polymer has therefore been demonstrated. The possible role of such interaction in the control of bacterial cell-wall synthesis is discussed. Of the other compounds investigated mono- and di-nucleotides were shown to be inhibitory, indicating that nucleotide pyrophosphorylase activities may be influenced by the energy charge of the cell.     

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.     

Evidence for early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells

Various mitogens activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent fibroblasts. Increased synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP) may precede or underlie these activations, but little direct evidence has been presented for this notion, due to lack of suitable analytical methods. To preferentially label intracellular ribose phosphate and quantitatively follow metabolic flux through PRPP into nucleotides, we prepared [ribosyl-14C]inosine and used it as a tracer. Evidence showed the validity of this method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure of PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that epidermal growth factor plus insulin stimulates the metabolic flux through PRPP. Similar extents of stimulation were induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone, suggesting the presence of an unknown signaling pathway common to these mitogens. This system is highly useful for studies of the mechanisms that stimulate in situ activity of PRPP synthetase.     

Synergistic effect of purine derivatives on the toxicity of pyrazofurin and 6-azauridine towards cultured mammalian cells

A novel synergistic effect of several purine derivatives such as adenine, adenosine, hypoxanthine, and guanine on the toxicity of nucleoside analogs pyrazofurin and 6-azauridine towards cultured Chinese hamster ovary (CHO) cells has been observed. The presence of the above purine derivatives enhanced the toxicity of pyrazofurin and 6-azauridine, in a dose dependent manner. The growth inhibitory effects of these nucleoside analogs either alone or in combination with the purine derivatives were reversed by uridine and cytidine, providing evidence that the synergistic effect of the purine derivatives was exerted at the level of pyrimidine nucleotide biosynthesis. Studies with mutant cells lacking various purine phosphorylating enzymes show that phosphorylation of purine derivatives through reactions utilizing phosphoribosylpyrophosate (PRPP) is essential for observing the synergistic response. It is suggested that the above purine derivatives (including adenosine, via conversion to hypoxanthine) exert their synergistic effects by depleting the cellular pool of PRPP by two separate mechanisms (direct utilization and feedback inhibition of its synthesis), which as a result becomes rate limiting in the synthesis of orotidine monophosphate (OMP). The reduced levels of OMP, which is a competing substrate with pyrazofurin- and 6-azauridine-5'-monophosphates for binding to the target enzyme OMP decarboxylase, could then account for the inhibition of the enzyme at lower concentrations of these analogs.     

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: an evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K_m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.     

Nucleotide and pentose synthesis after serum-stimulation of resting 3T6 fibroblasts.

We have investigated the de novo synthesis of intermediates of purine nucleotides in 3T6 fibroblasts and determined the manner by which the activity of this pathway is increased in resting cells by the addition of fresh serum. Within 30 minutes after stimulation, 3T6 cells began to synthesize increased amounts of purines by the de novo pathway as measured by increased amounts of formylglycinamide ribonucleotide, a representative intermediate of this pathway. Within 15 minutes after serum-stimulation 3T6 cells exhibited a substantial increase in their capacity to synthesize ribose compounds, particularly in the form of 5-phosphoribosylpyrophosphate (PRPP). The availability of PRPP appeared to be limiting for the synthesis of purine nucleotides in resting fibroblasts, but not necessarily in serum-stimulated cells. The amount of the enzyme PRPP synthetase as measured in vitro remained constant for at least the first four hours. Therefore, a study was made of various compounds known to activate PRPP synthetase in vitro. No evidence was found that suggested involvement of concentrations of cyclic nucleotides or phosphate. Experiments with methylene blue, an artificial electron acceptor that stimulates the production of ribose 5-phosphate by the hexose monophosphate shunt, indicated that one of the immediate consequences of the addition of serum is increased cycling of the pyridine nucleotide coenzymes, NADP⁺ and NADPH, and that the rapid increase in formation of ribose compounds and, consequently, purine nucleotides was caused as a result of modulation by this coenzyme. The relative ration of ATP:ADP:AMP as well as their concentrations remain constant in resting and serum-stimulated cells under normal assay conditions. However, there was a substantial decrease in ATP concentrations with a corresponding increase in AMP concentration with methylene blue in the assay buffer. The production of AMP from ATP was 5-fold greater in the serum-stimulated than in the resting fibroblasts. The increased production of AMP is thus serum-dependent and may reflect a basic enzymatic function of proliferative as compared to resting cells.     

Purine metabolism: determination of PRPP-amidotransferase and PRPP-synthetase in lymphocytes from patients with chronic lymphatic leukemia (CLL)

Phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase is the "key anabolic enzyme" of purine nucleotide synthesis; PRPP synthetase connects the pentose cycle with the same pathway. We have studied their behavior in 5 control subjects and in 8 affected by CLL. Determination of PRPP amidotransferase was carried out through the evaluation of 14C-glutamic acid (released by 14C-glutamine) in the incubation mixture. PRPP synthetase was followed by adding ATP and ribose 5-phosphate to the incubation mixtures, and by evaluating the PRPP formed through the release of CO2 in a coupled reaction. In the case of PRPP-amidotransferase, our values are in the range reported in the literature: in patients affected by CLL, the enzyme activity is much higher and the increase is more evident when values referred to the patients, than when to the cells. Our values of PRPP synthetase are consistent with those of Peters and Veerkamp, but no definite conclusion is possible in the case of leukemic patients.     

Studies on Metabolic Pathway of NAD in Yeast

Quantitative studies on yeast 5′-nucIeotidase are presented.

K_m values for purine 5′-nucleotides were generally smaller than those for pyrimidine 5′-nucleotides and, among purine series, K_m value for 5′-AMP was the smallest, while their V values were almost same.

The enzyme activity was inhibited in the competitive type by bases, nucleosides, 3′- or 2′-nucleotides, and NMN and in the mixed type by NAD and NADP.

Base-, ribose-, 3′- or 5′-phosphate moiety of nucleoside and nucleotide had some effects on binding with enzyme; especially the structure of base moiety characterizes the K_m or K_i value.

The enzyme activity was accelerated by Ni⁺⁺ or Co⁺⁺, which increases V value but never affects K_m value.

The relationship between the structure of substrate and its affinity towards enzyme is discussed.     

The adenine phosphoribosyltransferase from Giardia lamblia has a unique reaction mechanism and unusual substrate binding properties

Purine phosphoribosyltransferases catalyze the Mg2+ -dependent reaction that transforms a purine base into its corresponding nucleotide. They are present in a wide variety of organisms including plants, mammals, and parasitic protozoa. Giardia lamblia, the causative agent of giardiasis, lacks de novo purine biosynthesis and relies primarily on adenine and guanine phosphoribosyltransferases (APRTase and GPRTase) constituting two independent and essential purine salvage pathways. The APRTase from G. lamblia was cloned and expressed with a 6-His tag at its C terminus and purified to apparent homogeneity. Adenine and alpha-d-5-phosphoribosyl-1-pyrophosphate (PRPP) have K(m) values of 4.2 and 143 microm with a k(cat) of 2.8 s(-1) in the forward reaction, whereas AMP and PP(i) have K(m) values of 87 and 450 microm with a k(cat) of 9.5 x 10(-3) s(-1) in the reverse reaction. Product inhibition studies indicated that the forward reaction follows a random Bi Bi mechanism. Results from the kinetics of equilibrium isotope exchange further verified a random Bi Bi mechanism in the forward reaction. In a mutant enzyme, F25W, with kinetic constants similar to those of the wild type and a tryptophan residue at the adenine binding site, the addition of adenine or AMP to the free mutant enzyme resulted in fluorescence quenching, whereas PRPP caused fluorescence enhancement. The dissociation constants thus estimated are 16.5 microm for adenine, 14.3 microm for AMP, and 83.0 microm for PRPP. PP(i) exerted no detectable effect on the tryptophan fluorescence at all, suggesting a lack of PP(i) binding to the free enzyme. An ordered substrate binding in the reverse reaction with AMP bound first followed by PP(i) is thus postulated.     

The nucleotide sequence recognised by the Escherichia coli D type I restriction and modification enzyme.

A type I restriction endonuclease from a new isolate of Escherichia coli (E. coli E166) has been purified and characterised. The enzyme, EcoD, has a recognition sequence similar in overall structure to the previously determined type I enzyme sequences, an exception being that it is degenerate. The sequence is 5'-T-T-A-N-N-N-N-N-N-N-G-T-C-Y-3' 3'-A-A-T-N-N-N-N-N-N-N-C-A-G-R-5' where Y is a pyrimidine, R is a purine and N can be any nucleotide. The enzyme methylates adenosyl residues in both strands of the DNA that are separated by ten base pairs, suggesting that the enzyme interacts with DNA along one face of the helix making contacts in two successive major grooves.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

The c-Myc target gene Rcl (C6orf108) encodes a novel enzyme, deoxynucleoside 5'-monophosphate N-glycosidase

RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.     

Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase: Repression by End Products

Phosphoribosylpyrophosphate (PRPP) synthetase participates in the biosynthesis in bacteria of purine nucleotides, pyrimidine nucleotides, tryptophan, and histidine. The regulation of the synthesis of PRPP synthetase in Salmonella typhimurium was studied. Addition of end products to the growth medium, singly or in combination, resulted in small decreases in the specific activity of PRPP synthetase, but levels of the enzyme were never decreased to less than half of those found when the bacteria were grown on minimal medium. Growth of the bacteria on several different carbon sources or starvation for phosphate had little effect on the specific activity of PRPP synthetase. Over-production of histidine in a histidine regulatory mutant, which would be expected to result in a depletion of intracellular PRPP pools, did not alter PRPP synthetase specific activity. PRPP synthetase levels were examined in auxotrophic strains of S. typhimurium that had been starved for the end products of PRPP. In each case derepression of an enzyme in the biosynthetic pathway for the limiting end product was demonstrated. However, only alterations in the levels of pyrimidine bases in the culture medium brought about derepression and repression of PRPP synthetase. Excess pyrimidines do not completely repress the enzyme. Deprivation of exponentially growing cells for pyrimidines by growth of an auxotrophic mutant on media containing orotic acid, which enters the cells slowly, resulted in a 10-fold derepression of PRPP synthetase. Derepression of PRPP synthetase during uracil starvation was prevented by chloramphenicol. The PRPP synthetase activities of extracts from repressed and derepressed cells responded in identical fashion to heat inactivation, cellulose acetate electrophoresis at several pH values, and in kinetic experiments.     

The Sites of Action of Allopurinol in Crithidia fasciculata*

Reversal of the growth inhibition of Crithidia fasciculata by allopurinol requires both a purine and a pyrimidine. Hypoxanthine is the most effective purine in the reversal. Cell-free extracts were prepared which were capable of the decarboxylation of orotidine 5′-phosphate. Other enzyme preparations carried out the phosphoribosylation of allopurinol. By the use of [4-¹⁴C] orotidine 5′-phosphate (enzymatically prepared), it was shown that allopurinol ribotide (enzymatically prepared), but not the free base, inhibits orotidine 5′-phosphate decarboxylase.