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Endogenous ligands acting on a human P2Y12 receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y12 ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca2+ assay using the CHO cells stably expressing P2Y12-G16alpha fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y12 receptor as agonists with the EC50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y12, P2Y13, P2Y14, GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions.

CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-a-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992-->Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954-->Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch-Nyhan syndrome

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.

Regulation of the purine biosynthetic gene purA was examined by using a transcriptional fusion to a luciferase reporter gene. Transcription was repressed about 10-fold by the addition of adenine and increased approximately 4.5-fold by the addition of guanosine. This regulation is mediated by a purine repressor (PurR). In a purR mutant, basal expression was increased 10-fold, and there was no further stimulation by guanosine or repression by adenine. An open reading frame, yabJ, immediately downstream from purR was found to have a role in the repression of purA by adenine. Repression by adenine was perturbed in a purR+ yabJ mutant, although guanosine regulation was retained. Mutations in the PurR PRPP binding motif abolished guanosine regulation in the yabJ mutant. Thus, PRPP appears to be required for upregulation by guanosine. The amino acid sequence of YabJ is homologous to the YER057c/YjgF protein family of unknown function.

Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold. The enzyme catalyzes the Mg-dependent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP) to yield AMP. The purification procedure included anion exchange chromatography, chromatofocusing and gel filtration. Elution of the enzyme from the chromatofocusing column indicated a pI value of 4.7. The molecular mass for the native enzyme was 50 kDa; however, upon electrophoresis under denaturing conditions two bands of apparent molecular mass of 29 and 20 kDa were observed. We have previously reported the presence of two separate coding sequences for APRT, APT1 and APT2 in S. cerevisiae. The appearance of two bands under denaturing conditions suggests that, unlike other APRTs, this enzyme could form heterodimers. This may be the basis for substrate specificity differences between this enzyme and other APRTs. Substrate kinetics and product inhibition patterns are consistent with a ping-pong mechanism. The Km for adenine and PRPP were 6 microM and 15 microM, respectively and the Vmax was 15 micromol/min. These kinetic constants are comparable to the constants of APRT from other organisms.

Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl-alpha-1-diphosphate (PPRibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing K(m) for PPRibP by about fivefold without affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PPRibP displayed constant velocities. This indicated that PPRibP and GTP influenced the structure of the enzyme. Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PPRibP. Guanosine-3',5'-bis(diphosphate) was found to activate the enzyme much like GTP.

Labeling of human and schistosomal hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) with GMP-2',3'-dialdehyde (ox-GMP) results in nearly complete inactivation of the enzymes. Digestion of the [3H]ox-GMP-modified HGPRTases with trypsin followed by high-performance liquid chromatographic fractionation, partial amino acid sequencing, and mass spectral analysis of the labeled peptides revealed that four peptides from each of the two HGPRTases were labeled with ox-GMP. The conclusion from these studies indicates that two segments of the human enzyme protein, Ser 4-Arg 47 and Ser 91-Arg 100, and one region in the schistosomal enzyme, Gly 95-Lys 133, were labeled by ox-GMP. Since the ox-GMP labeling of human HGPRTase was effectively blocked by either GMP or PRibPP, whereas that of schistosomal HGPRTase was inhibited only by GMP [Kanaaneh, J., Craig, S. P., III, & Wang, C. C. (1994) Eur. J. Biochem. 223, 595-601], the two labeled peptides in human enzyme may be involved in binding to both GMP and PRibPP while the one peptide in schistosomal enzyme may be implicated only in GMP binding. We have also confirmed a previous observation [Keough, D. T., Emmerson, B. T., & de Jersey, J. (1991) Biochim. Biophys. Acta 1096, 95-100] that carboxymethylation of Cys 22 in the human HGPRTase by iodoacetate was inhibited by PRibPP. We also demonstrated that the carboxymethylation of Cys 25 in schistosomal HGPRTase by iodoacetate was specifically blocked by PRibPP. Apparently, the N-terminal regions in both enzymes are involved in PRibPP binding. The fact that ox-GMP labels the N-terminal region in human enzyme but not in schistosomal enzyme and that PRibPP protects against ox-GMP labeling in human enzyme but not in schistosomal enzyme both suggest that the amino-terminal PRibPP-binding site may be in close proximity to the GMP-binding site in human HGPRTase but not in schistosomal HGPRTase. This clear distinction between the active sites of human and schistosomal HGPRTases could be further exploited for potential opportunities for antischistosomal chemotherapy.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-alpha-pyrophosphoryl-2-alpha, 3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate (cPRPP). Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation. First, cPRPP was not able to "activate" Cys1 for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 for catalysis. Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site. However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site. Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme.

Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.

The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of human and the parasitic trematode, Schistosoma mansoni, were expressed at high levels in transformed Escherichia coli in their native forms. Guanosine 2',3'-dialdehyde 5'-phosphate (ox-GMP) was shown to bind irreversibly to both enzymes in a time-dependent manner. This binding was stabilized by sodium borohydride reduction, suggesting that a Schiff's base is formed between the dialdehyde groups of ox-GMP and the amino group of a lysine residue in the enzymes. This linkage formation applies also to inosine 2',3'-dialdehyde 5'-phosphate but not to adenosine 2',3'-dialdehyde 5'-phosphate. GMP was found to be protective against ox-GMP inactivation and [3H]ox-GMP labeling of both HGPRTases. 5-Phosphoribosyl-1-diphosphate (PRibPP) also protects human HGPRTase against the ox-GMP inactivation and [3H]ox-GMP labeling but provides virtually no protection against the ox-GMP inactivation and labeling of the schistosomal enzyme, even though PRibPP binds to the latter with a threefold higher affinity. These results imply that PRibPP and ox-GMP compete with each other for binding to the human HGPRTase but not for binding to the schistosomal enzyme. This discrepancy could be exploited for the purpose of designing selective inhibitors of the schistosomal HGPRTase. Guanosine 2',3'-dialdehyde (ox-guanosine) is nearly as active as ox-GMP in inhibiting schistosomal HGPRTase but much less potent in inhibiting human HGPRTase, suggesting that ox-guanosine and ox-GMP may bind equally well to the parasite enzyme. PRibPP can protect human but not schistosomal HGPRTase against the inactivation by ox-guanosine. Therefore, ox-GMP and ox-guanosine must be forming Schiff's bases with the same amino acid residues in each of the two HGPRTases.

Glutamine phosphoribosylpyrophosphate amidotransferase, the key regulatory enzyme for de novo purine nucleotide synthesis, is subject to feedback regulation by adenine and guanine nucleotides. Affinity labeling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-monophosphate (N3-AMP) was used to identify purine nucleotide sites for feedback control of the Escherichia coli amidotransferase. FSBA inactivated the amidotransferase with saturation kinetics. Specificity for inactivation was shown by the covalent attachment of 2.0-2.4 eq of [3H] sulfobenzoyladenosine (SBA) per subunit and protection by GMP and AMP against inactivation and incorporation of [3H]SBA. Six chymotryptic peptides modified with [3H]SBA were isolated and identified by differential labeling followed by high performance liquid chromatography and radioactivity. Mass spectrometry and Edman degradation analysis were used to identify 5 residues that were covalently modified by [3H]SBA: Tyr74, Tyr258, Lys326, Tyr329, and Tyr465. Tyr258 was also modified by N3-AMP. Mutant enzymes K326Q and Y329A had activity similar to that of the wild type enzyme. However, both mutants exhibited decreased sensitivity to inhibition by GMP and decreased binding of GMP but were inhibited by AMP. Mutant enzymes Y74A and Y258F were normally feedback-inhibited but were defective in glutamine amide transfer and synthase functions, respectively. Therefore Tyr74 and Tyr258 are important for activity and modification by FSBA and N3-AMP accounts for enzyme inactivation. These results localize residues important for catalysis in close proximity to a site for nucleotide binding. Two additional mutant enzymes, G331I and N351A, were constructed which were refractory to inhibition by GMP with little change in inhibition by AMP. A replacement of Tyr465 indicates that this residue is not essential for catalysis or feedback inhibition. Overall, these results are interpreted in terms of a two-nucleotide site model with Lys326, Tyr329, Gly331, and Asn351 defining a site required for inhibition by GMP. A second nucleotide site not affinity labeled by analogs is very close to or overlaps with the catalytic site.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).

Bovine liver glutamate dehydrogenase (GDH) is a very complex enzyme in its molecular structure and activity regulation. The present paper deals with the effect of PRPP on GDH activity. This phosphate sugar, playing a key role in purine metabolism, behaves as an effector in the L-glutamate oxidation, showing both an activating effect (at low L-glu concentration) and an inhibiting one (as far as 40%), increasing with L-glu and coenzyme (3-acetyl-pyridine adenine dinucleotide) concentration. The inhibition is quite evident at low PRPP concentration, thus suggesting a physiologic role in cellular regulation of GDH activity.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.

A system of hypoxanthine uptake and IMP retention was studied and characterized in human erythrocytes. It follows closely the system already described for rabbit erythrocytes[7]. IMP formation and retention are dependent on the activity of hypoxanthine phosphoribosyl-transferase and on intracellular availability of phosphoribosyl pyrophosphate (P-Rib-PP), which is one of the substrates. In the extrecellular medium, neither P-Rib-PP nor GMP -- a potent inhibitor of the enzyme in vitro -- has any influence on IMP retention. The amount of residual hypoxanthine phosphoribosyltransferase in erythrocyte ghost preparations is directly related to the residual hemoglobin content. Thus the enzyme is characterized as typically soluble and "loosely bound" to membranes. There is a slight difference in the kinetic properties of the ghost-bound and the free soluble enzyme. The possible importance of these results for purine uptake and utilization in human red cells is discussed.

The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase.

The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation-and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenase. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30-40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools inthe hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of the dGTP pool. The concentrations of dATP and dCTP also declined, but these alterations were less pronounced and the pools returned to normal after 2 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.

The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with lysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine.

The metabolism of 5-fluorouracil (5-FU) in tumors and normal tissues of humans was investigated in vitro. Phosphorylation of 5-FU was faster in tumor tissues than in normal tissues. Phosphorylating activity with 2-deoxy-alpha-D-ribose 1-phosphate (dRiblP) and ATP as cofactors was more active than that with alpha D-ribose 1-phosphate (RiblP) and ATP, or 5-phospho-alpha-D-ribosyldiphosphate (PPRibP) as cofactors. Phosphorylating activity in squamous cell carcinoma of the lung was similar to that in adenocarcinomas. Degradation of 5-FU was much faster in the liver than in other tissues including tumor tissues.

The effects of polyamines were studied on carbamoyl-phosphate synthase II (EC 6.3.5.5.) which is the first and rate limiting enzyme in mammalian pyrimidine synthesis. Polyamines in physiological concentrations (0.1-1 mM) strongly inhibited the carbamoyl-phosphate synthesis. Of the polyamines tested spermine was the most effective followed by spermidine and putrescine. Spermine increased the KM for ATP and the requirement for Mg++ of carbamoyl-phosphate synthase reaction. UTP, an inhibitor, had similar, while phosphoribosyl-pyrophosphate, an activator of the enzyme had an opposite effect. Increasing concentrations of phosphoribosyl-pyrophosphate completely reversed the inhibition caused by spermine, while did not influence the degree of inhibition caused by UTP. A possible physiological role of polyamines in synchronizing the substrate and activator functions of phosphoribosyl-pyrophosphate in pyrimidine synthesis is suggested.

Studies were done to examine direct binding of the first enzyme of the histidine biosynthetic pathway (phosphoribosyltransferase) to 32P-labeled phi80dhis DNA and competition of this binding by unlabeled homologous DNA and by various preparations of unlabeled heterologous DNA, including that from a defective phi80 bacteriophage carrying the histidine operon with a deletion of part of its operator region. Our findings show that phosphoribosyltransferase binds specifically to site in or near the regulatory region of the histidine operon. The stability of the complex formed by interaction of the enzyme with the DNA was markedly decreased by the substrates of the enzyme and was slightly increased by the allosteric inhibitor, histidine. These findings are consistent with previous data that indicate that phosphoribosyltransferase plays a role in regulating expression of the histidine operon.

The rate of the conversion of ribose 5-phosphate to hexose 6-phosphates by reaction of the non-oxidative pentose phosphate pathway was measured in the presence of various biological materials. Of 22 compounds tested, PRPP and ADP markedly inhibited the formation of hexose 6-phosphates from ribose 5-phosphate. The transketolase activity in beef liver enzyme preparation was extremely inhibited by PRPP and ADP, but the transaldolase activity was not inhibited. The mode of inhibition of transketolase by PRPP and ADP was a competitive one. The Ki value for PRPP was 0.14 mM and that for ADP 0.54 mM with respect to transketolase. We discuss the possible regulatory roles of ADP and PRPP on pentose phosphate metabolism in the pentose phosphate pathway.

The activity of some enzymes involved in pyrimidine nucleotide synthesis was studied in rat embryo cell (REC) cultures infected with X14 or H-1 parvovirus. dCMP aminohydrolase activity of the infected cells was 64--121% greater than that of the mock-infected cells. dTMP synthetase activity was 18% greater in X14 virus-infected cells and 34% lower in H-1 virus-infected cells. These results suggest some differences in the infected cells, as regards the biosynthesis of dTMP. Orotate phosphoribosyltransferase and orotidylate decarboxylase activities appeared nearly unmodified compared to the mock-infected cells. The addition of phosphoribosylpyrophosphate (PRPP) to the cell suspension incubated with [6-14C] orotate increased the specific radioactivity of acid-soluble uracil, 5-fold in the mock-infected cells and 15- --24-fold in the X14 or H-1 virus-infected cells (72 hr p.i.). This result suggests that the lowered pyrimidine nucleotide synthesis in infected cells depends to a large extent on the diminished PRPP pool.

Allosteric activation of pyruvate kinase from a thermophilic bacterium, Bacillus stearothermophilus, by ribose 5-phosphate (R5P) was kinetically examined. Two activated forms of this enzyme could be distinguished, depending on the R5P concentration. One form (Form I) was observed at about 10(-5) M R5P. It showed a slightly negative cooperativity for phosphoenolpyruvate (PEP). The other form (Form II) was observed at more than 10(-3) M R5P and showed Michaelis-Menten kinetics for PEP. The PEP and ADP concentrations that yield half-maximal velocity were essentially identical for the two forms (about 0.1 and about 0.5 mM, respectively), but Form I had a larger Vmax value than Form II. In the absence of R5P, the enzyme showed a homotropic positive cooperativity for PEP; the concentration required for the half-maximal velocity was about 2 mM and that of ADP was about 1.6 mM. The enzyme was more susceptible to protease digestion in the presence of R5P than in the absence of it. The concentration of R5P required for the enzyme to be susceptible to protease digestion was approximately identical with that required to generate Form I. With more than 10(-3) M R5P, the thermostability of the enzyme was greatly increased. The concentration of R5P required for the enzyme to be thermostable was in good agreement with that required to generate Form II. These results indicate that the two activated forms distinguished kinetically differ in their conformations, too. The saturating level of PEP did not cause such a change in the thermostability or the susceptibility to protease.

After mutations affecting the de novo adenine nucleotide synthesis in Bacillus anthracis, the salvage pathway failed to ensure growth at the prototroph level. In sonicated suspensions of adenyl succinate lyase deficient B. anthracis mutant, in the presence of exogenous 5-phosphoribosyl-1-pyrophosphate (PRPP), part of the labelled adenine was incorporated into adenine nucleotides. Addition of PRPP to cultures of adenyl succinate lyase and adenyl succinate synthetase mutants failed to promote growth. Both kinds of auxotroph showed in vitro a reduced growth rate as compared to the prototrophic strain.

Amidophosphoribosyltransferase (EC 2.4.2.14) has been partially purified from pigeon liver, and ligand-induced alterations in molecular and kinetic properties have been studied. In Tris-HCl buffer the predominant form of the enzyme has an s20,w of 5.9 +/- 0.7, Stokes radium of 42 A, and estimated molecular weight of 102,000. Incubation with phosphoribosylpyrophosphate (PP-ribose-P) results in an increase in the s20,w to 7.9 +/- 0.6, Stokes radius to 53 A, and estimated molecular weight to 172,000. Incubation of this larger form with purine ribonucleotides leads to a decrease in the molecular weight of amidophosphoribosyltransferase that is proportional to the concentration of purine ribonucleotide. Purine ribonucleotides produce sigmoidal kinetics with respect to the substrate PP-ribose-P, with Hill coefficients of 1.4 to 1.6 and 1.8 to 2.0 in the presence of AMP and GMP, respectively. Incubation with 0.6 M KCl leads to sigmoidal kinetics. Hill coefficient of 1.8 and dissociation of the larger form of amidophosphoribosyltransferase. Inorganic phosphate has complex effects upon the enzyme. In 25 mM potassium phosphate buffer the enzyme aggregates to a large form with an s20,w of 8.3 +/- 0.2, Stokes radius of 53 A, and estimated molecular weight of 181,000. Inorganic phosphate and PP-ribose-P both stabilize the enzyme to storage in vitro at 4 degrees. However, inorganic phosphate is 4 times more effective than PP-ribose-P in preventing inactivation of the enzyme by sodium dodecyl sulfate. Inorganic phosphate produces sigmoidal kinetics with respect to PP-ribose-P, Hill coefficient of 1.5. The interaction coefficients for AMP and GMP are reduced from 1.8 to 1.2 and 2.2 to 1.4, respectively, in the presence of 25 mM potassium phosphate. It is concluded that pigeon liver amidophosphoribosyltransferase is a complex allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.

Phosphate-dependent glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) from rat liver was found to be strongly activated by phosphoribosylpyrophosphate (P-rib-PP), the substrate of amidophosphoribosyltransferase (EC 2.4.2.14). Since the assay of the latter is based on the P-rib-PP-dependent conversion of glutamine to glutamate, the amidotransferase activities determined in crude tissue preparations were found to be too high. The interference of glutaminase, however, could be completely eliminated by its inactivation at 50 degrees C. Amidotransferase was not affected by the heat treatment. Because of the increased rate of the glutamate formation at this temperature, the incubation time of the assay could be significantly reduced.

The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM).

1. At the lowered concentrations of 0.5 mM ATP and 1.5 mM MgCl2, 2.0 mM UTP, UDP and UMP inhibited the activity of Crithidia fasciculata carbamoyl-phosphate synthetase II by about 65, 80 and 40% respectively. 2. The result suggests that feedback inhibition of the activity by uridine nucleotides is a mechanism of regulation of the de novo pyrimidine biosynthetic pathway in C. fasciculata. 3. ADP, AMP and CDP inhibited the activity (about 70, 40 and 40%). 4. Excess Mg2+ at around 1 mM, relative to the ATP concentration, was required for the maximum activity. 5. 5-Phosphoribosyl 1-pyrophosphate had no significant effect on the activity under various conditions examined.

Carbamylphosphate synthetase was purified to homogeneity from a derepressed strain of Salmonella typhimurium by a procedure based on affinity chromatography employing immobilized glutamine. The enzyme catalyzes the synthesis of carbamylphosphate from either ammonia or glutamine together with ATP and bicarbonate. The ATP saturation curve of either nitrogen donor is sigmoidal (n equals 1.5) but the affinity for ATP is higher with ammonia. In addition to the feedback inhibition by UMP and activation by ornithine which we previously reported (1), the activity was found to be stimulated by IMP and phosphoribosyl-1-pyrophosphate. Evidence from pool measurements in enteric bacteria by others suggests that of the latter two compounds only phosphoribosyl-1-pyrophosphate is physiologically significant. All effectors regulate enzyme activity by altering its affinity for ATP. Glutamine also modulates the affinity for ATP; it is increased as glutamine concentratiions decrease, an effect that could serve to insulate the cell against major changes in carbamylphosphate synthesis in response to fluctuations in concentration of glutamine. The molecular weight of the holoenzyme was estimated to be 150,000 by sucrose density gradient centrifugation in triethanolamine and Tris-acetate buffers in which the enzyme is a monomer. In the presence of ornithine in potassium phosphate buffer, the enzyme is an oligomer with a molecular weight of 580,000. This transition has been exploited as an alternate route of purifying the enzyme to homogeneity using successive sucrose density centrifugation. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate shows that the enzyme consists of two unequal subunits with molecular weights of 110,000 and 45,000. The two subunits were separated by gel filtration in the presence of 1 M potassium thiocyanate, ATP, MgCl2, glutamine, NH4Cl, ornithine, and UMP. The heavy subunit catalyzes the synthesis of carbamylphosphate from ammonia but not glutamine. The ATP saturation curve for the separated heavy subunit is still sigmoidal (n equals 1.4 and So.5 equals 0.3 mM). The ammonia dependent activity of the heavy subunit is stimulated by the activators ornithine, IMP, and phosphoribosyl-1-pyrophosphate but is only marginally inhibited by high concentrations of UMP. The addition of the light subunit restored full ability to utilize glutamine as well as normal sensitivity to UMP. Purified subunits were used for in vitro complementation studies with strains carrying mutations in pyrA, the structural gene encoding carbamylphosphate synthetase. The results indicate that the pyrA region encodes both subunits and that the structural genes for the two polypeptides are linked. A deletion mutant lacking both subunits of carbamylphosphate synthetase also lacked any ability to synthetize carbamylphosphate from ammonia. Hence, unlike certain other bacteria, S. typhimurium does not possess a carbamate kinase.