The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110

For the derivation of an inosine-overproducing strain from the wild type microorganism, it is known that the addition of an adenine requirement, removal of purine nucleoside hydrolyzing activity, removal of the feedback inhibition, and repression of key enzymes in the purine nucleotides biosynthetic pathway are essential. Thus, the disruption of purA (adenine requirement), deoD (removal of purine nucleosides phosphorylase activity), purR (derepression of the regulation of purine nucleotides biosynthetic pathway), and the insensitivity of the feedback inhibition of phosphoribosylpyrophosphate (PRPP) amidotransferase by adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) were done in the Escherichia coli strain W3110, and then the inosine productivity was estimated. In the case of using a plasmid harboring the PRPP amidotransferase gene (purF) that encoded a desensitized PRPP amidotransferase, purF disrupted mutants were used as the host strains. It was found that the innovation of the four genotypes brought about a small amount of inosine accumulation. Furthermore, an adenine auxotrophic mutant of E. coli showed inappropriate adenine use because its growth could not respond efficiently to the concentration of adenine added. As the presence of adenosine deaminase is well known in E. coli and it is thought to be involved in adenine use, a mutant disrupted adenosine deaminase gene (add) was constructed and tested. The mutant, which is deficient in purF, purA, deoD, purR, and add genes, and harboring the desensitized purF as a plasmid, accumulated about 1 g of inosine per liter. Although we investigated the effects of purR disruption and purF gene improvement, unexpectedly an increase in the inosine productivity could not be found with this mutant.     

Antifolates induce primary inhibition of the de novo purine pathway prior to 5-aminoimidazole-4-carboxamide ribotide transformylase in leukemia cells.

Polyglutamated dihydrofolate, accumulated as a result of potent inhibition of dihydrofolate reductase (DHFR), has been postulated to directly inhibit the purine pathway at 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (reaction 9) in leukemia cells exposed to methotrexate (MTX). We have observed that 25 microM MTX or piritrexim, a "non-classical" antifolate, induce several-fold accumulations of AICAR and N-succino-AICAR to a combined cellular concentration of 89 microM in mouse L1210 leukemia cells after 2 h. By contrast, complete inhibition of reaction 4 by 25 microM azaserine results in accumulation of N-formyl-glycinamide ribotide (FGAR) polyphosphates to a combined cellular concentration of greater than 10 mM. MTX prevented azaserine-induced accumulation of FGAR polyphosphates. Hence, these antifolates induce primary inhibition of the de novo purine pathway at, or prior to, glycinamide ribotide transformylase (reaction 3).     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant

Summary We have studied the relationship between purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt ^- gpt^-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt ^-) while the second class represented purine regulatory mutants (purR ^-). There is thus selection for the purR phenotype in a hpt ^- gpt^-background.Abbreviations FGAR formyl glycinamide ribotide - HPRT hypoxanthine phosphoribosyl transferase - GPRT guanine phosphoribosyl transferase - APRT adenine phosphoribosyl transferase - PRPP 5 phosphoribosyl-1 pyrophosphate - 6MP 6-mercaptopurine - FA 2-fluoroadenine     

Glutamine phosphoribosylpyrophosphate amidotransferase-independent phosphoribosyl amine synthesis from ribose 5-phosphate and glutamine or asparagine

Phosphoribosylamine (PRA) is the first intermediate in the common pathway to purines and thiamine and is generated in bacteria by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (EC 2.4.2.14) from PRPP and glutamine. Genetic data have indicated that multiple, non-PRPP amidotransferase mechanisms exist to generate PRA sufficient for thiamine but not purine synthesis. Here we describe the purification and identification of an activity (present in both Escherichia coli and Salmonella enterica) that synthesizes PRA from ribose 5-phosphate and glutamine/asparagine. A purification resulting in greater than a 625-fold increase in specific activity identified 8 candidate proteins. Of the candidates, overexpression of AphA (EC 3.1.3.2), a periplasmic class B nonspecific acid phosphatase, significantly increased activity in partially purified extracts. Native purification of AphA to >95% homogeneity determined that the periplasmic l-asparaginase II, AnsB (EC 3.5.1.1), co-purified with AphA and was also necessary for PRA formation. The potential physiological relevance of AphA and AnsB in contributing to thiamine biosynthesis in vivo is discussed.     

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.     

Regulation of de novo purine biosynthesis in Chinese hamster cells

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.     

A Simple and Rapid Method for the Separation and Characterization of Hypoxanthine-Guanine Phosphoribosyltransferase Isoenzymes

Purinephosphoribosyltransferases catalyze the conversion of purine bases to their nucleotides in the presence of 5-phosphoribosyl-l- pyrophosphate (PRPP) (1). This salvage pathway plays an important role in the regulation of de novo purine synthesis (2). In mammalian cells two distinct phosphoribosyltransferases were demonstrated: the enzyme adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyl- transferase; A-PRT; E.C. 2.4.2.7) and the enzyme hypoxanthine-guanine phosphoribosyltransferase (PIP: pyrophosphate phosphoribosyltranferase; HG-PRT; E.C. 2.4.2.8). There has been a great interest in this latter enzyme as the complete absence of this enzyme activity in patients with Lesch-Nyhan syndrome (3) and a partial deficiency in some patients with gout (4) has been demonstrated.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin fibroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

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.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

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.     

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.     

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 A resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 A resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 A resolution. Comparisons of these three hAPRT structures with other 'type I' PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPP. Comparative analyses presented here provide structural evidence to propose the role of Glu104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.     

Modified pathway to synthesize ribulose 1,5-bisphosphate in methanogenic archaea

Several sequencing projects unexpectedly uncovered the presence of genes that encode ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) in anaerobic archaea. RubisCO is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway, a scheme that does not appear to contribute greatly, if at all, to net CO2 assimilation in these organisms. Recombinant forms of the archaeal enzymes do, however, catalyze a bona fide RuBP-dependent CO2 fixation reaction, and it was recently shown that Methanocaldococcus (Methanococcus) jannaschii and other anaerobic archaea synthesize catalytically active RubisCO in vivo. To complete the CBB pathway, there is a need for an enzyme, i.e., phosphoribulokinase (PRK), to catalyze the formation of RuBP, the substrate for the RubisCO reaction. Homology searches, as well as direct enzymatic assays with M. jannaschii, failed to reveal the presence of PRK. The apparent lack of PRK raised the possibility that either there is an alternative pathway to generate RuBP or RubisCO might use an alternative substrate in vivo. In the present study, direct enzymatic assays performed with alternative substrates and extracts of M. jannsachii provided evidence for a previously uncharacterized pathway for RuBP synthesis from 5-phospho-D-ribose-1-pyrophosphate (PRPP) in M. jannaschii and other methanogenic archaea. Proteins and genes involved in the catalytic conversion of PRPP to RuBP were identified in M. jannaschii (Mj0601) and Methanosarcina acetivorans (Ma2851), and recombinant Ma2851 was active in extracts of Escherichia coli. Thus, in this work we identified a novel means to synthesize the CO2 acceptor and substrate for RubisCO in the absence of a detectable kinase, such as PRK. We suggest that the conversion of PRPP to RuBP might be an evolutional link between purine recycling pathways and the CBB scheme.     

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.     

产嘌呤核苷的枯草杆菌prsA基因在大肠杆菌中的表达

为了克隆产嘌呤核苷的枯草杆菌prsA基因。用PCR扩增的方法,从产肌苷的枯草杆菌Bacillus subtilis JSIM-1019中克隆出一个长1kb长的DNA片段,经功能检测,证明正向插入片段与大肠杆菌的磷酸核糖焦磷酸营养缺陷特性（PREP-）能够营养互补。含有该重组质粒的PRPP缺陷大肠杆菌JSIM—DH-27在基本培养基上的能够生长。     

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.     

Overproduction disease in man due to enzyme feedback resistance mutation. Purine overproduction in gout due to excessive activity of mutant feedback-resistant phosphoribosylpyrophosphate synthetase.

Physiologically superactive phosphoribosylpyrophosphate (PRPP) synthetase, due to feedback resistance mutation, was found in a family with excessive purine production, gout and uric acid lithiasis. The superactivity of the mutant enzyme was manifest in the propositus' erythrocytes and cultured fibroblasts, in increased generation, content and metabolic availability of PRPP, leading in the fibroblasts to acceleration of the rate of purine synthesis de novo. One of the propositus' two siblings was similarly affected, but the propositus' father, his second brother and four sons, were all clinically and biochemically normal. The mother was clinically normal and normouricemic, but hyperuricosuric. Cultured fibroblasts from her skin exhibited variability in PRPP content and availability and in the rate of purine synthesis de novo. The mother's cultures were found to contain a mosaicism of two cell populations, one with normal and the other with mutant PRPP synthetase, indicating an X-linked pattern of inheritance of the PRPP synthetase abnormality in this gouty family.