Orotidinuria induced by allopurinol.

The administration of allopurinol to patients suffering from hyperuricemia and to normal subjects results in increased excretion of the pyrimidine nucleoside orotidine. Evidence is presented for the interference by allopurinol of the de novo biosynthesis of uridine 5'-phosphate in man.

Studies on the coordinate activity and liability of orotidylate phosphoribosyltransferase and decarboxylase in human erythrocytes, and the effects of allopurinol administration.

A coordinate relationship between the activities of two sequential enzymes in the de novo pyrimidine biosynthetic pathway has been demonstrated in human red cells. The two enzymes, orotidylate phosphoribosyltransferase and decarboxylase are responsible for the conversion of orotic acid to uridine-5'-monophosphate. Fractionation of red cells, on the basis of increase of specific gravity with cell age, has revealed that these two enzymes have a marked but equal degree of lability in the ageing red cell. It is postulated that orotidylate phosphoribosyltransferase and decarboxylase form an enzyme-enzyme complex, and that the sequential deficiency of these two enzymes in hereditary orotic aciduria may reflect a structural abnormality in this complex. In patients receiving allopurinol, the activities of both enzymes are coordinately increased, and this increase appears to be due, at least in part, to stabilization of both orotidylate phosphoribosyltransferase and decarboxylase in the ageing red cell. Allopurinol ribonucleotide is an in vitro inhibitor of orotidine-5'-monophosphate decarboxylase and requires the enzyme hypoxanthineguanine phosphoribosyltransferase for its synthesis. However, the administration of allopurinol to patients lacking this enzyme results in orotidinuria and these patients have elevated orotidylate phosphoribosyltransferase and decarboxylase activities in their erythrocytes. Evidence is presented that the chief metabolite of allopurinol, oxipurinol, with a 2,4-diketo pyrimidine ring is capable of acting as an analogue of orotic acid. It is postulated that the in vivo formation of oxipurinol ribonucleotide, catalyzed by orotidylate phosphoribosyltransferase, after allopurinol administration, leads to inhibition of orotidine-5'-monophosphate decarboxylase. This inhibition results in the urinary excretion of excessive amounts of orotidine and orotic acid, and "pseudo-substrate" stabilization of orotidylate phosphoribosyltransferase and decarboxylase.

Improved procedures for the synthesis of phosphomevalonate and for the assay and purification of pig liver phosphomevalonate kinase.

An improved procedure for the synthesis of phosphomevalonate using excess free ATP4-, and phenyl agarose to remove contaminating nucleotides, is described. A high-voltage electrophoresis assay, which separates phosphomevalonate from mevalonate 5-diphosphate at pH 3.5, was developed for the assay of phosphomevalonate kinase (ATP:5-phosphomevalonate phosphotransferase, EC 2.7.4.2). High-voltage electrophoresis, at pH 5, could also be used for the separation of mevalonate 5-diphosphate from isopentenyl diphosphate. An alternative method for the purification of phosphomevalonate kinase from pig liver was also developed. The high-voltage electrophoresis assay was used to reassess the metal ion and nucleotide specificity of the pig liver phosphomevalonate kinase. ATP could be partially replaced by ITP and GTP and poorly by CTP and UTP. Apparent activation of the enzyme by free ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224).

Phosphorothioate analogues of adenosine triphosphate as substrates of the mevalonate kinase reaction.

Adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma S]) and the diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP[alpha S]) and adenosine 5'-O-(2-thiotriphosphate) (ATP[beta S]) could act as substrates for mevalonate kinase (ATP: mevalonate 5-phosphotransferase, EC 2.7.1.36) in the presence of different activating divalent metal cations. In the presence of Mg2+ the Rp diastereomers of both ATP[alpha S] and ATP[beta S] were the preferred substrates, whereas in the presence of Cd2+ the Sp diastereomers were the more active. Calculation of Vmax/Km values confirmed reversal of the Rp/Sp ratios for ATP[beta S] with Mg2+ [16] and Cd2+ [2], providing evidence that the metal is coordinated to the beta-phosphate at the active site. However, the corresponding ratios were very similar for ATP[alpha S], 0.4 and 0.2 for Mg2+ and Cd2+, respectively, so that it is unlikely that the metal ion was coordinated to the alpha-phosphate, in the active complex. The Vmax for CdATP [gamma S] was some 90-fold less than that for MgATP[gamma S], suggesting that Cd2+ is bound to S from the gamma-P and that the breaking of the Cd-S bond is the rate determining step of the reaction. It was concluded that the chelate structure of metal ATP substrate in the mevalonate kinase reaction is the lambda,beta,gamma-bidentate MgATP chelate. The probable product of the reaction is delta,alpha,beta-bidentate MgADP.

An improved purification procedure, an alternative assay and activation of mevalonate kinase by ATP.

An improved procedure for the purification of pig liver mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) is described. A high-voltage electrophoresis assay was developed for mevalonate kinase. The procedure separates mevalonate from phosphomevalonate and also from diphosphomevalonate so that it can be used to measure the subsequent enzyme, phosphomevalonate kinase (EC 2.7.4.2). The assay has allowed the reassessment of the metal ion and nucleotide specificity of the pig liver enzyme. Some of the previously reported properties reflected those of the enzymes in the coupling assay rather than mevalonate kinase itself. A series of compounds were tested as activators or inhibitors of mevalonate kinase. It was found that ATP4-, arsenate and, to a smaller extent, inorganic phosphate activated the enzyme. At fixed MgATP2- (1 mM) concentrations the activation of mevalonate kinase by free ATP4- at pH 8.0 was observed at concentrations at up to 10-fold that of MgATP2- before causing any inhibition. The presence of free ATP4- resulted in a biphasic Lineweaver-Burke plot with apparent Km values for MgATP2- being 0.14 mM and 60 microM, respectively. Fluorescence measurements were consistent with the notion that the binding of excess ATP4- to the enzyme caused a conformational change.

Molecular cloning and characterization of the Plasmodium falciparum cytidine triphosphate synthetase gene.

Using degenerate oligonucleotides derived from conserved amino acid regions of cytidine triphosphate synthetase, a fragment of the gene from the malarial parasite, Plasmodium falciparum, was isolated by polymerase chain reaction (PCR). This fragment was used as a probe in the isolation of genomic clones containing the entire pfCTP synthetase coding region (2580 bp). The gene encodes the largest CTP synthetase found in any organism to date due to the presence of two additional sequences which are part of the continuous open reading frame and are not introns as their presence in the mRNA was confirmed by reverse transcriptase-PCR. These features distinguish the parasite enzyme from that of the host making it an attractive target for structure based drug design.

Studies on pig liver mevalonate-5-diphosphate decarboxylase.

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.

31P-NMR study of the orotate phosphoribosyltransferase equilibrium with thiopyrophosphate as substrate.

31P-nuclear magnetic resonance spectroscopy was used to directly determine the equilibrium of the reaction catalysed by yeast orotate phosphoribosyltransferase, using orotidine monophosphate and inorganic pyrophosphate as substrates. A Keq value of 0.71 was determined, in good agreement with that of 0.49 calculated by Victor, Greenberg and Sloan (J. Biol. Chem. 254 (1979) 2647-2655), from kinetic data. Substitution of thiopyrophosphate as the substrate shifted the position of the equilibrium 55-fold, to yield a Keq value of 39. Only the beta S analogue of 5-phosphoribosyl 1-diphosphate appeared to be synthesized in this reaction.

Effect of monoclonal anti-neuraminidase antibodies on the kinetic behavior of influenza virus neuraminidase.

Neuraminidase from the recombinant influenza virus A/NWSHA-Tokyo/3/67NA HON2 has been shown to exhibit non-Michaelis-Menten kinetics. The multiphasic behaviour was demonstrated for both the isolated neuraminidase heads and for the intact virus. Interaction of the enzyme with two monoclonal anti-neuraminidase antibodies (WANA 1 and RANA 1), which recognize separate antigenic determinants on the molecule, resulted in hyperbolic kinetic behaviour. While both antibodies abolished the multiphasic kinetics of the enzymic reaction, only WANA 1 altered the Vmax and Km values, indicating that it may in some way inhibit the interaction of enzyme and substrate.

Enzymes of pyrimidine biosynthesis in Crithidia luciliae.

Evidence has been obtained for the presence of enzymes of both the de novo and salvage pyrimidine pathways in the protozoan parasite, Crithidia luciliae. Carbamyl phosphate synthetase-II activity could not be unequivocally demonstrated in crude extracts. However, a distinct peak of activity with a molecular weight of approximately 500 000 was observed following chromatography on Sepharose CL-6B. The enzyme preferentially utilised glutamine with respect to ammonia. It was inhibited by UTP and 5-phosphoribosyl-1-diphosphate had a small activating effect. Carbamyl phosphate synthesis by a 'phosphorolytic' citrullinase could not be demonstrated. The ensuing three de novo enzymes could also be separated on Sepharose CL-6B. Approximate molecular weights were estimated: aspartate transcarbamylase (150,000); dihydroorotase (90,000) and dihydroorotate dehydrogenase (70,000). As reported previously, orotate phosphoribosyltransferase and orotidylate decarboxylase were particulate, being associated with the glucosome. Activities of the salvage enzymes, uracil phosphoribosyltransferase, uridine phosphorylase and uridine nucleosidase were observed. All enzymes were cytoplasmic. No uridine kinase activity was detected.

Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline.

The activity of Plasmodium falciparum dihydroorotate dehydrogenase, a particulate, electron transport-linked enzyme involved in de novo pyrimidine synthesis, was depressed when the parasite was cultured in the presence of a therapeutic concentration of tetracycline over a 96 h period. There was no direct inhibitory effect of the antibiotic on the enzyme activity. The activity of glutamate dehydrogenase, which is cytoplasmic in the parasite, was unaffected by tetracycline over the same period. Dihydroorotate dehydrogenase activity was substantially recovered when electron acceptors were added. It is suggested that the effect of tetracycline is manifested at the level of the dehydrogenase and/or the electron transport chain linked to this enzyme.

Serine hydroxymethyltransferase from pyrimethamine-sensitive and -resistant strains of Plasmodium chabaudi.

Serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified from a pyrimethamine sensitive strain of Plasmodium chabaudi. Km values of 2.91 and 1.08 mM were determined for tetrahydrofolate and serine, respectively. The effects of pH, of temperature and of some potential inhibitors were determined. The enzyme was also partially purified from a pyrimethamine-resistant strain of P. chabaudi and subjected to the same regime. No differences between the enzymes from the two sources could be detected. It would appear that the changes in properties in the enzymes dihydrofolate reductase and thymidylate synthetase associated with the development of drug resistance in P. chabaudi were not reflected in any obvious alterations in serine hydroxymethyltransferase.

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo.

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23).

Isolation, characterization and expression of the gene encoding cytidine triphosphate synthetase from Giardia intestinalis.

The cytidine triphosphate synthetase gene from Giardia intestinalis was cloned using a PCR-based strategy. A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and KTKPTQ. This product was used to probe restriction endonuclease digested genomic DNA and the respective plasmid mini-libraries. Two genomic clones were obtained one with a 3.6 kb HindIII DNA fragment, containing approximately three-quarters of the 5'-end of the synthetase gene and subsequently, a 5.8 kb PstI DNA fragment which contained the whole gene. The intronless gene has a 1863 bp open reading frame encoding 620 amino acids (M(r) of 68.3 kDa). A well conserved catalytic glutamine aminotransferase (GAT) domain was identified. In addition, three insert sequences were found which are not present in CTP synthetase from other species. Alignment and comparison of the deduced amino acid sequence relative to CTP synthetases from other species revealed a high degree of identity (34%) with a greater resemblance to prokaryotes than eukaryotes. The gene is located on chromosome 6 and the messenger RNA encoding it is estimated to be 1.9 kb. The coding region of G. intestinalis CTP synthetase was generated by PCR and subsequently cloned into the pQE30 vector for expression in E. coli. This construct yielded a soluble and enzymatically active recombinant protein which was purified by a Ni-NTA affinity column. The purified recombinant protein had a subunit molecular weight of 69.5 kDa and a native molecular weight of approximately 274 kDa. Kinetic studies of the partially purified recombinant G. intestinalis CTP synthetase gave apparent K(m) values of 0.1 mM and approximately 0.5 mM for the substrates UTP and L-glutamine respectively in accord with previously reported values for the native enzyme.

Deoxynucleoside kinases of Giardia intestinalis.

Giardia intestinalis lacks the ability to synthesise deoxyribonucleotides de novo and must rely on salvage synthesis. Two separate kinases, specific for purines (deoxyadenosine and deoxyguanosine) and pyrimidines (thymidine and deoxycytidine), respectively, are responsible for the incorporation of deoxyribonucleosides. A substantial degree of purification was achieved for the purine deoxynucleoside kinase by the combination of Mono Q anion exchange chromatography, preparative gel electrophoresis and Superose 12 gel filtration. An overall recovery of 4%, with 186- and 174-fold purification, for deoxyguanosine kinase and deoxyadenosine kinase activities, respectively, was observed. The molecular weight was found to be approximately 80,000 by gel filtration. Only a partial purification of thymidine/deoxycytidine kinase was achieved. However, both pyrimidine activities remained associated throughout various purification procedures and appeared to be associated with a protein of 44 kDa.

Purification and characterization of uridine (thymidine) phosphorylase from Giardia lamblia.

Giardia lamblia is totally dependent on salvage synthesis for its pyrimidine requirements. The salvage pathway enzyme, uridine phosphorylase (pyrimidine nucleoside phosphorylase) was purified to apparent homogeneity from G. lamblia crude extracts by fast protein liquid chromatography and gel filtration on a Superose 12 column, resulting in an overall 3500 fold purification and a recovery of 7.5%. Mono P chromatofocusing gave rise to a major activity peak eluting from the column at pH 5.9, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight was found to be 43,000 +/- 2000 from the Superose 12 column, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000 +/- 2000, indicating that it is a monomer. The activities of uridine, deoxyuridine and thymidine phosphorylases from G. lamblia remained associated throughout the purification procedure, suggesting that one enzyme is responsible for the three enzyme activities. The ratio of activities was consistent throughout the purification procedure. In the reverse (anabolic) direction, the enzyme could use both uracil and thymine as substrates. The properties of the phosphorylase differ significantly from those of the mammalian host.

Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii.

All six enzymes of the de novo biosynthetic pathway leading to the biosynthesis of UMP have been characterized in Toxoplasma gondii. The first three enzymes of the pathway, carbamyl phosphate synthetase-II (CPS-II), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) could be consistently separated by sucrose gradient centrifugation. Their molecular weights were estimated to be approximately 540 000, 140 000 and 70 000, respectively. The last two enzymes, orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase), cosedimented at the same position, corresponding also to a molecular weight of approximately 70 000. The fourth enzyme, dihydroorotate dehydrogenase (DHO-DHase), was associated with the particulate fraction. Apparent Km values for the respective enzymes were: CPS-II, MgATP2- (19.7 1.2 mM), L-glutamine (12.0 +/- 1.7 microM), ammonia (15.5 +/- 2.7 mM); ATCase, carbamyl phosphate (26.2 +/- 3.5 microM), L-aspartate (17.6 +/- 8.5 mM); DHOase (reverse direction) dihydroorotate (1.6 +/- 0.08 microM); ODCase, orotidine 5'-monophosphate (0.41 +/- 0.04 microM). MgUTP2- was found to act as an inhibitor of CPS-II, with an apparent Ki of 0.41 mM. However, 5-phospho-alpha-D-ribosyl-1-diphosphate, dimethyl sulphoxide and glycerol had no effect on the Km value for MgATP2-. The effect of some inhibitors, including pyrimidine and purine nucleotides and analogs and respiratory chain inhibitors, was also determined for the enzymes of the pathway.

Metabolic studies of the protozoan parasite, Crithidia luciliae, using proton nuclear magnetic resonance spectroscopy.

Proton nuclear magnetic resonance (NMR) spectroscopy was used to follow glucose metabolism in Crithidia luciliae. Parasites were grown aerobically and anaerobically in culture, with glucose as the major carbon source and 1H NMR spectra were acquired for the cell free medium. The 1H NMR resonances of metabolites utilised and produced during cell growth were identified by difference spectroscopy, and quantitated from standard curves using 3-trimethylsilyl propionate-2,2,3,3-d4 sodium salt as an internal standard. The major metabolites produced by C. luciliae grown aerobically on 8 mM glucose were succinate, pyruvate, acetate and ethanol, in final concentrations in the media when the cells entered stationary phase of 8.5 +/- 0.5, 5.0 +/- 0.3, 2.1 +/- 0.2 and 2.5 +/- 0.6 mM, respectively. The production of succinate and pyruvate, but not acetate and ethanol, followed closely the growth curve of the parasites. Succinate was also measured enzymically and glucose using an autoanalyser. In both cases the results correlated well with the NMR data. The amounts of end products formed were greater than could be accounted for by the utilisation of glucose or any other metabolite observable in the 1H NMR spectra. There was approximately one extra atom of carbon for each molecule of succinate formed, supporting the view that succinate is produced via phosphoenolpyruvate carboxykinase and carbon dioxide fixation. Anaerobically the same major metabolites were produced, but with a decreased ratio of succinate to acetate and ethanol. The formation of glycerol from glucose was not observed under these conditions.

Arginine metabolism during culture of Giardia intestinalis.

The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined. Supplementation of the normal growth medium (Diamond's TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days. There was a corresponding rapid utilisation of arginine, with none being detectable after this time. The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway. Subsequently, there was a decrease in the ammonia concentration in the medium. Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth. In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia. However, in these conditions, the ammonia concentration remained elevated. This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent. The observations confirm the operation of a functional arginine dihydrolase pathway in G. intestinalis. The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth. It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamond's TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.

Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites.

The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.