The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties

Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyl-transferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyl-transferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [¹⁴C]hypoxanthine, [¹⁴C]guanine or [¹⁴C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 μM, 3.8 μM and 52.4 μM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodiumfalciparum, which are dimers, with xanthine about 100-times less proficient as a substrate. T. foetus HGXPRTase is thus a unique enzyme with opportunity for specific inhibitor design.     

Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasite's host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.     

Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

An amperometric biosensor for fish freshness detection from xanthine oxidase immobilized in polypyrrole-polyvinylsulphonate film

A new amperometric biosensor was developed for determining hypoxanthine in fish meat. Xanthine oxidase with pyrrole and polyvinylsulphonate was immobilized on the surface of a platinum electrode by electropolymerization. The determination of xanthine-hypoxanthine was performed by means of oxidation of uric acid liberated during the enzyme reaction on the surface of the enzyme electrode at + 0.30V (SCE). The effects of pH, substrate concentration, and temperature on the response of the xanthine-hypoxanthine biosensor were investigated. The linear working range of the enzyme electrode was 1.0 × 10(-7) -1.0 × 10(-3) M of the hypoxanthine concentration, and the detection limit was 1.0 × 10(-7)M. The apparent K(m(app)) and I(max) of the immobilized xanthine oxidase were found to be 0.0154 mM and 1.203 μA/mM, respectively. The best pH and temperature value for xanthine oxidase were selected as 7.75 and 25°C, respectively. The sensor was used for the determination of hypoxhantine in fish meat. Results show that the fish degraded very rapidly after seven days and the hypoxanthine amount was found to increase over days of storage.     

The trypanocidal Cape buffalo serum protein is xanthine oxidase.

Plasma and serum from Cape buffalo (Syncerus caffer) kill bloodstream stages of all species of African trypanosomes in vitro. The trypanocidal serum component was isolated by sequential chromatography on hydroxylapatite, protein A-G, Mono Q, and Superose 12. The purified trypanocidal protein had a molecular mass of 150 kDa, and activity correlated with the presence of a 146-kDa polypeptide detected upon reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences of three peptide fragments of the 146-kDa reduced polypeptide, ligand affinity and immunoaffinity chromatography of the native protein, and sensitivity to pharmacological inhibitors, identified the trypanocidal material as xanthine oxidase (EC 1.1.3.22). Trypanocidal activity resulted in the inhibition of trypanosome glycolysis and was due to H2O2 produced during catabolism of extracellular xanthine and hypoxanthine by the purine catabolic enzyme.     

Xanthine and hypoxanthine in amniotic fluid during pregnancy

Xanthine and hypoxanthine are indicators of cellular hypoxia. The purpose of our study was to establish a reference range for the concentrations of xanthine and hypoxanthine in amniotic fluid. Of a total of 38 included in the investigation, 27 pregnant women were found to have low values for xanthine (less than 1.2 mumol/l) and hypoxanthine (less than 0.4 mumol/l). To our knowledge, this is the first attempt to define a reference range for xanthine and hypoxanthine concentration in amniotic fluid during pregnancy.     

The multicopper oxidase of Mycobacterium tuberculosis (MmcO) exhibits ferroxidase activity and scavenges reactive oxygen species in activated THP-1 cells

The MmcO protein of Mycobacterium tuberculosis is a membrane-associated multicopper oxidase. Its natural substrate(s) and its role in pathogenesis are not well characterized. A recent report proposes that MmcO contributes to copper resistance in M. tuberculosis during infection. We have expressed and reconstituted the active enzyme from inclusion bodies in E. coli. MmcO exhibits maximal activity against the experimental substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) or ABTS, at pH 4. The enzyme also exhibits ferroxidase activity at pH 4. Most notable was the finding that MmcO is able to scavenge the reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase enzyme system. This ROS scavenging activity of MmcO was also evident against ROS generated by THP-1 cells. We propose that MmcO protects M. tuberculosis during infection against ROS attack in addition to providing copper resistance to the pathogen.     

利用DNA免疫法制备植物选择标记基因hpt表达蛋白抗体的研究

目的：制备转基因作物中选择标记基因——潮霉素B磷酸转移酶(hygromycin B phosphotransferase，hpt)基因表达产物的多克隆抗体，并探讨影响核酸免疫效果的因素及相应机制。方法：以hpt的cDNA全长插入真核表达载体pcDNA3中，并经限制性内切酶酶切及DNA测序鉴定。以纯化的重组质粒pCDNA3-HPT免疫BALB／c小鼠。用在E．coli中表达并纯化的(His)6—HPT进行ELISA，检测免疫过程中小鼠血清抗体效价的增长状况，并用Western blot检测抗血清的特异性。结果：经3次核酸免疫后，小鼠血清中未发现明显的抗HPT抗体一第4次加强免疫时，将小鼠分为3组，每组两只。第1组改用去内毒素的质粒提取试剂盒提纯的重组质粒免疫，第2组用(His)6-HPT融合蛋白免疫，第3组仍用原来提取的重组质粒免疫。结果发现，第1组小鼠抗血清的效价有所上升，经ELISA检测达1：200；第2组抗血清的滴度显著升高，达到1：2000；而第3组小鼠血清中仍未检测到明显的抗体：Western blot证实，前两组抗血清均可与亲和层析纯化的GST—HPT、(His)6-HPT融合蛋白及其诱导表达的相应菌体总蛋白发生特异性结合。结论：用DNA免疫法成功地制备了抗hpt基因表达产物的特异性抗体，但抗体效价不够理想。推测与hpt基因本身的性质及其在体内表达呈现的水平有关，可望通过州整影响核酸免疫的其他多种因素提高抗体的水平。     

Purine metabolizing enzymes of Plasmodium lophurae and its host cell, the duckling (Anas domesticus) erythrocyte

Adenosine kinase, adenosine deaminase, hypoxanthine phosphoribosyltransferase, inosine-nucleoside phosphorylase, 5'-AMP deaminase and 5'-IMP nucleotidase were identified in cell-free extracts of duckling erythrocytes; no evidence for 5'-AMP nucleotidase and xanthine oxidase activity was found. The Km values for the duckling red cell enzymes were similar to those reported for human erythrocytes. Plasmodium lophurae extracts demonstrated similar enzyme activities except for 5'-AMP deaminase and 5'-IMP nucleotidase which were absent. It is proposed that during infection erythrocytic AMP is catabolized to IMP, inosine and hypoxanthine; the hypoxanthine is taken up by the plasmodium, utilized to form IMP, and this in turn is converted into adenine and guanine nucleotides.     

Purification of hypoxanthine-guanine phosphoribosyltransferase of Plasmodium lophurae

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was isolated from the malarial parasite, Plasmodium lophurae. The apparent pI, as determined by chromatofocusing, was 7.6. The native molecular weight was 79,000. The pH profile of HGPRT exhibited a broad pH optimum. With hypoxanthine as substrate maximal activity was achieved from pH 6.0-10.0, and with guanine as substrate maximal activity occurred from pH 7.5-9.5. The enzyme exhibited Michaelis-Menten kinetics with all substrates. The Km values were 3.8 microM (hypoxanthine), 2.4 microM (guanine), 6.2 microM (6-mercaptopurine), 7.6 microM (6-thioguanine), and 360 microM (8-azahypoxanthine). 6-Thioinosine, 9-beta-arabinofuranosylhypoxanthine, 6-chloropurine, xanthine and azaguanine were inhibitors of the P. lophurae enzyme. From the substrate and inhibitor data it appears that the sixth position on the purine ring plays a major role in enzyme activity.     

Cellular expression of xanthine oxidoreductase protein in normal human tissues.

Xanthine oxidoreductase is an important cytoplasmic source of reactive oxygen species, and has been implicated in the pathogenesis of ischemia-reperfusion damage. Because the cellular localization of this protein remains unclear, our aim was to study its distribution in fresh normal human tissue obtained at surgery. For immunohistochemical studies we purified the protein from human milk and raised a polyclonal antibody in rabbits. In the liver the protein was preferentially localized to the periportal hepatocytes and it was absent from the perivenous region. In the proximal intestine, the protein was expressed in epithelial cells and goblet cells. Lactating mammary gland acinar cells showed intense staining. Small vessel vascular endothelial cells of the intestine, mammary gland, and skeletal muscle showed immunoreactivity, but in the kidney, glomerular endothelial cells were negative. No cells in the heart, brain, or lung expressed the enzyme protein. The observed localization of the xanthine oxidoreductase protein is consistent with previously observed enzyme activities in the organs studied. The widely assumed exclusive localization to capillary endothelium obviously does not apply to humans.     

Effect of propionyl carnitine on energy charge and adenine nucleotide content of cardiac endothelial cells during hypoxia

Adenine mucleotide metabolism is very active in endothelial cells. These cells are very rich in xanthine oxidase which may produce oxygen reactive species during ischaemia and reperfusion when a high amount of adenine nucleotides may be catabolized to hypoxanthine. We investigated the effect of propionyl carnitine on energy charge and nucleotide content in cultured endothelial cells during changes in oxygen partial pressure. During hypoxia the adenine nucleotide pool and the energy charge decreased more slowly in the presence of 0.5 mM propionyl carnitine than in the absence of the compound. Furthermore during reoxygenation a more rapid increase of energy charge and adenine nucleotide concentration was observed with propionyl carnitine. These observations suggest that the presence of propionyl carnitine allows the endothelial cells to maintain their functionality and regulatory role on vessel activity for a longer time and decreases the formation of oxygen reactive species due to xanthine oxidase activity on hypoxanthine formed by adenine nucleotide catabolism.     

Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme.

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.     

Changes in plasma hypoxanthine and free radical markers during exercise in man

Eight men cycled for about 6 minutes at workloads corresponding to 44 and 72% of maximal oxygen uptake and to fatigue at 98% maximal oxygen uptake. Blood samples from a brachial artery and a femoral vein were taken at rest and during exercise. Hypoxanthine, xanthine and urate in plasma were significantly elevated at fatigue and after 10 minutes of recovery. Only hypoxanthine showed a significant arterio-femoral venous difference. The release of hypoxanthine from the legs increased during the recovery period and was three-fold higher 10 minutes post exercise than at the end of exercise. It is concluded that the marked increase in plasma hypoxanthine which occurs during intensive exercise originates from the working muscle whereas the transformation to xanthine and urate may occur in other tissues. Glutathione, methemoglobin and malondialdehyd (MDA) were used as plasma markers of free radicals. Total glutathione (glutathione + glutathionedisulfide) in blood and plasma increased during intensive exercise and may be indicative of free radical formation. However, MDA was not detectable in plasma during any conditions (less than 0.1 mumol x l-1 plasma) and methemoglobin decreased slightly during exercise. Further studies using more specific techniques are required to determine whether the formation of free radicals is increased after brief intensive exercise.     

Determination of urinary methylated purine pattern by high-performance liquid chromatography

We describe the group selective separation and quantification of unmodified and modified purines in human urine by high-performance reverse phase liquid chromatography. The pattern of oxypurines and methylated purines: hypoxanthine (Hx), xanthine (X), 1-methyl hypoxanthine (1-MHx), 1-methyl guanine (1-MG), 3-methyl guanine (3-MG), 7-methyl guanine (7-MG), 1-methyl xanthine (1-MX), 3-methyl xanthine (3-MX), 7-methyl xanthine (7-MX), 1,7-dimethyl guanine (1,7-dMG), 1,3-dimethyl xanthine (1,3-dMX), 1,7-dimethyl xanthine (3,7-dMX) and 1,3,7-trimethyl xanthine (1,3,7-tMX) were determined in a single run in urine of a healthy subject and a gout patient before and after treatment with allopurinol. This method may be useful to investigate the urinary pattern of methylated bases in diseases involving purine metabolism.     

Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I

Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.     

Is ZMP the toxic metabolite in Lesch–Nyhan disease?

The genetic deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), located on the X chromosome, causes a severe neurological disorder in man, known as Lesch-Nyhan disease (LND). The enzyme HPRT is part of the savage pathway of purine biosynthesis and catalyzes the conversion of hypoxanthine and guanine to their respective nucleotides, IMP and GMP. HPRT deficiency is associated with a relatively selective dysfunction of brain dopamine systems. Several metabolites that accumulate in the patients (phosphoribosylpyrophosphate (PRPP), hypoxanthine, guanine, xanthine, and Z-nucleotides) have been proposed as toxic agents in LND. Some authors have pointed that Z-riboside, derived from the accumulation of ZMP, could be the toxic metabolite in LND. However, the available experimental data support a better hypothesis. I suggest that ZMP (and not Z-riboside) is the key toxic metabolite in LND. ZMP is an inhibitor of the bifunctional enzyme adenylosuccinate lyase, and a deficiency of this enzyme causes psychomotor and mental retardation in humans. Moreover, it has been reported that ZMP inhibits mitochondrial oxidative phosphorylation and induces apoptosis in certain cell types. ZMP is also an activator of the AMP-activated protein kinase (AMPK), a homeostatic regulator of energy levels in the cell. The AMPK has been implicated in the regulation of cell viability, catecholamine biosynthesis and cell structure. I propose that accumulation of ZMP will induce a pleiotropic effect in the brain by (1) a direct inhibition of mitochondrial respiration and the bifunctional enzyme adenylosuccinate lyase, and (2) a sustained activation of the AMPK which in turns would reduce cell viability, decrease dopamine synthesis, and alters cell morphology. In addition, a mechanism to explain the accumulation of ZMP in LND is presented. The knowledge of the toxic metabolite, and the way it acts, would help to design a better therapy.     

Human adenylosuccinate lyase (ADSL), cloning and characterization of full-length cDNA and its isoform, gene structure and molecular basis for ADSL deficiency in six patients

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.     

Fibrinogen Cleavage by the Streptococcus pyogenes Extracellular Cysteine Protease and Generation of Antibodies That Inhibit Enzyme Proteolytic Activity

The extracellular cysteine protease from Streptococcus pyogenes is a virulence factor that plays a significant role in host-pathogen interaction. Streptococcal protease is expressed as an inactive 40-kDa precursor that is autocatalytically converted into a 28-kDa mature (active) enzyme. Replacement of the single cysteine residue involved in formation of the enzyme active site with serine (C192S mutation) abolished detectable proteolytic activity and eliminated autocatalytic processing of zymogen to the mature form. In the present study, we investigated activity of the wild-type (wt) streptococcal protease toward human fibrinogen and bovine casein. The former is involved in blood coagulation, wound healing, and other aspects of hemostasis. Treatment with streptococcal protease resulted in degradation of the COOH-terminal region of fibrinogen alpha chain, indicating that fibrinogen may serve as an important substrate for this enzyme during the course of human infection. Polyclonal antibodies generated against recombinant 40- and 28-kDa (r40- and r28-kDa) forms of the C192S streptococcal protease mutant exhibited high enzyme-linked immunosorbent assay titers but demonstrated different inhibition activities toward proteolytic action of the wt enzyme. Activity of the wt protease was readily inhibited when the reaction was carried out in the presence of antibodies generated against r28-kDa C192S mutant. Antibodies produced against r40-kDa C192S mutant had no significant effect on proteolysis. These data suggest that the presence of the NH(2)-terminal prosegment prevents generation of functionally active antibodies and indicate that inhibition activity of antibodies most likely depends on their ability to bind the active-site region epitope(s) of the protein.