Purine-metabolizing enzymes in Babesia divergens

Extracts of Babesia divergens were examined for the enzymes which catalyse purine salvage. Adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), inosine phosphorylase (EC 2.4.2.1), purine phosphoribosyltransferases (EC 2.4.2.7, EC 2.4.2.8, EC 2.4.2.22) and nucleoside kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73) were all detected at relatively high activities, whereas nucleotide interconverting enzymes were not detected. Coformycin and 4-amino-5-imidazolecarboxamide were found to be potent inhibitors of adenosine deaminase and guanine deaminase, respectively. The results suggest that B. divergens is capable of synthesizing purine nucleotides via two routes, one involving purine phosphoribosyltransferases and the other employing nucleoside kinases.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.     

Adenosine is the primary precursor of all purine nucleotides in Trichomonas vaginalis

Trichomonas vaginalis, a parasitic protozoan and the causative agent of trichomoniasis, lacks de novo purine nucleotide synthesis and possesses a unique purine salvage pathway, consisting of a bacterial type purine nucleoside phosphorylase and a purine nucleoside kinase. It is generally believed that adenine and guanine are converted to their corresponding nucleosides and then further phosphorylated to form AMP and GMP, respectively, as the main as well as the essential pathway of replenishing the purine nucleotide pool in the organism. Formycin A, an analogue of adenosine, inhibits both enzymes as well as the in vitro growth of T. vaginalis with an estimated IC(50) of 0.27 microM. This growth inhibition was reversed by adding adenine to the culture medium but not by adding guanine or hypoxanthine. Furthermore, T. vaginalis can grow in semi-defined medium supplemented with only adenine but not with guanine or hypoxanthine. Radiolabeling experiments followed by HPLC analysis of the purine nucleotide pool in T. vaginalis demonstrated incorporation of [8-14C]adenine into both adenine and guanine nucleotides, whereas [8-14C]guanine was incorporated only into guanine nucleotides. Substantial adenosine deaminase activity and significant IMP dehydrogenase and GMP synthetase activities were identified in T. vaginalis lysate, suggesting a pathway capable of converting adenine to GMP via adenosine. This purine salvage scheme depicts adenosine the primary precursor of the entire purine nucleotide pool in T. vaginalis and the purine nucleoside kinase one of the most pivotal enzymes in purine salvage and a potential target for anti-trichomoniasis chemotherapy.     

Isolation and characterization of a mutant Chinese hamster cell line resistant to the glutamine analog 6-diazo-5-oxo-l-norleucine

A mutant cell line, called don801, was isolated from a wild-type population of V79.5 Chinese hamster cells by its ability to grow in the presence of the glutamine analog 6-diazo-5-oxo-l-norleucine (DON), which is toxic for V79.5 cells. The don801 cells were found not to be cross-resistant to another glutamine analog, O-diazoacetyl-l-serine (azaserine, AS). It was shown that guanine but neither hypoxanthine nor adenine protected V79.5 cells from the toxic effects of DON, while hypoxanthine and adenine, but not guanine protected them against AS toxicity. Exposure of wild-type cells to DON was shown to result in a specific reduction of intracellular GTP pools, while in the mutant cells there was no effect on GTP levels. These results strongly suggested that DON was specifically inhibiting guanylate synthetase (GMP synthetase;xanthosine-5-phosphate:l-glutamine amidoligase, EC 6.3.5.2) in V79.5 cells and that the enzyme in don801 cells was resistant to inhibition. In vitro assays of GMP synthetase activities from V79.5 and don801 cells confirmed this hypothesis. The mutant phenotype was also found to be dominant in intraspecific cell hybrids.     

Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

Exogenous guanine was found to be incorporated into the nucleic acids of Chlamydia psittaci when the parasite was grown in HeLa cells containing hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) activity but not when the parasite was grown in transferase-deficient HeLa cells. No evidence for a chlamydia-specific transferase activity was found in either transferase-containing or transferase-deficient infected HeLa cells. It is concluded that C. psittaci is incapable of metabolizing guanine, but that the parasite can use host-generated guanine nucleotides as precursors for nucleic acid synthesis.     

The purine nucleotide cycle and its molecular defects.

Three enzymes of purine metabolism, adenylosuccinate synthetase, adenylosuccinate lyase and AMP deaminase, have been proposed to form a functional unit, termed the purine nucleotide cycle. This cycle converts AMP into IMP and reconverts IMP into AMP via adenylosuccinate, thereby producing NH3 and forming fumarate from aspartate. In muscle, the purine nucleotide cycle has been shown to function during intense exercise; the metabolic flux through the cycle has been proposed to play a role in the regeneration of ATP by pulling the adenylate kinase reaction in the direction of formation of ATP, and by providing Krebs cycle intermediates. In kidney, the purine nucleotide cycle was shown to account for the release of NH3 under the normal acid-base status, but not under acidotic conditions. In brain, the purine nucleotide cycle might function under conditions that induce a loss of ATP, and thereby contribute to its recovery. There is no evidence that the purine nucleotide cycle operates in liver. Deficiency of muscle AMP deaminase is an apparently frequent disorder, which might affect approximately 2% of the general population. The observation that it can be found in clinically asymptomatic individuals suggests, paradoxically, that the ATP-regenerating function which has been attributed to the purine nucleotide cycle is not essential for muscle function. Further work should be aimed at identifying the conditions under which AMP deaminase deficiency becomes symptomatic. Adenylosuccinate lyase deficiency provokes psychomotor retardation, often accompanied by autistic features. Its clinical heterogeneity justifies systematic screening in patients with unexplained mental deficiency. Additional studies are required to determine the mechanisms whereby this enzyme defect results in psychomotor retardation.     

Folate utilisation by Leishmania species and the identification of intracellular derivatives and folate-metabolising enzymes

The use and metabolism of folates by leishmanias have been studied by assessing the growth of promastigotes in defined media with different folates and the cell content of folate-metabolising enzymes. The folates present in Leishmania mexicana mexicana have been determined using HPLC. Folic acid, 5-formyltetrahydrofolate (THF) and 5-methyl-THF each supported growth of L. m. mexicana promastigotes in defined medium, whereas the parasites did not survive in the absence of folates; p-aminobenzoic acid could not replace the folate requirement. The only folate present at detectable levels in L. m. mexicana promastigotes was 5-methyl-THF. Dihydrofolate reductase (EC 1.5.1.3), methylene-THF reductase (EC 1.1.1.68), serine hydroxymethyltransferase (EC 2.1.2.1) and thymidylate synthetase (EC 2.1.1.45) were all detected in extracts of promastigotes of L. m. mexicana, L. donovani and L. major. Some of these activities were also found in extracts of amastigotes of the former two species. The enzymes of L. m. mexicana have been partially characterised. Methylene-THF reductase may be involved in the conversion in vivo of 5-methyl-THF to 5,10-methylene-THF.     

Guanosine deaminase in human serum and tissue extracts--a reappraisal of the products

Of the human salvage enzymes that deaminate ribonucleosides, two--cytidine deaminase and adenosine deaminase--have been found particularly useful for diagnostic purposes. In humans, no enzymes are present that can directly deaminate the bases of these ribonucleosides. Indeed, the only enzyme present that can directly deaminate a base is guanine deaminase, and the diagnostic usefulness of this enzyme has been well documented. The aim of this study is to identify the origin of the ammonia formed when human sera and tissue extracts are incubated with buffered guanosine, and to clarify whether the ammonia comes from the deamination of guanosine by guanosine deaminase or is produced as a result of deamination of guanine formed as a breakdown product of guanosine by purine nucleoside phosphorylase (PNP). Apparent deamination of guanosine by guanosine deaminase in human sera and tissue extracts was found to be due to two enzymes acting in tandem when the products of the reaction were examined by HPLC. The ribose was first removed from guanosine by PNP to form guanine, which was then deaminated to xanthine by guanine deaminase.     

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.     

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.     

Molecular analysis of HPRT deficiencies: an update of the spectrum of Asian mutations with novel mutations

Inherited mutations of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8), give rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in Asian patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the PCR technique coupled with direct sequencing. In this study, we update the spectrum of mutations with nine novel mutations. Two missense mutations (T124P and D185G) were detected in patients with HRH (HPRT-related hyperuricemia). In a patient having a severe partial deficiency of HPRT with neurological dysfunction (HRND: HPRT-related neurological dysfunction), a single nucleotide substitution (27+5G > A) causing a splicing error was found in intron 1. The mutation resulted in a remarkably decreased level of normal mRNA, and production of an abnormal mRNA with a 49-bp insert at the 5'-end of intron 1, which caused the frame-shift of an amino acid codon (10fs27X). In six typical Lesch-Nyhan families, we found two 3-bp deletions responsible for single amino acid deletions (V8del and Y28del), two 1-bp deletions (440delA and 635delG) generating a frame-shift, an insertion of two amino acids (159insKV), and a 4,131-bp deletion from introns 4 to 6 resulting in two types of abnormal mRNA. Including these nine mutations, 42 HPRT1 mutations have been identified among 47 Asian families with deficiency of HPRT.     

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.     

Enzymatic activities leading to pyrimidine nucleotide biosynthesis from cell-free extracts of Rickettsia typhi.

Cell-free extracts from Rickettsia typhi were examined for the presence or absence of pyrimidine phosphotransferase enzymes and compared with the enzymes of mouse L cells and Salmonella typhimurium. The organisms were grown in mouse L cells and in the yolk sacs of chicken embryos, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The enzymes for the reutilization of uridine and thymidine, uridine kinase (EC 2.7.1.48) and thymidine kinase (EC 2.7.1.21), were not detected in R. typhi extracts with the phosphate donors effective for control enzymes. The following enzyme activities were demonstrated in R. typhi: uridine-5'-monophosphate kinase (UMPK, EC 2.7.4.4), deoxythymidine-5'-monophosphate kinase (dTMPK, EC 2.7.4.9), and nucleosidediphosphate kinase (NDPK, EC 2.7.4.6). Physicochemical and enzymatic analyses demonstrated that the pyrimidine nucleotide kinases of R. typhi were not of host origin and that the source (yolk sac and mouse L cells) did not influence the relative enzymatic activities. The specific activities of UMPK and dTMPK were higher when the rickettsiae were harvested before embryo death, whereas NDPK levels were slightly decreased. The specific activities of UMPK, dTMPK, and NDPK were comparable to those of S. typhimurium, and consequently the rickettsiae have potential for the anabolism of monophosphates, as do the host-independent bacteria. These results suggest that R. typhi cannot utilize host uridine or thymidine pools directly but must rely on themonophosphorylated molecules of the host cell or must synthesize the monophosphates de novo.     

The Control of cell proliferation by preformed purines: A genetic study I. Isolation and preliminary characterization of Chinese hamster lines with single or multiple defects in purine “salvage” pathways

Sublines with single or multiple defects in purine salvage enzymes were isolated from the Chinese hamster fibroblastic line GMA32 through single or successive onestep selections for resistance to purine analogs. They were examined for their ability to incorporate purine bases and nucleosides into macromolecules, for their sensitivity to growth inhibitory purines, and for their rescue by exogenous purines from deprivation imposed by metabolic inhibitors of endogenous synthesis. The results show that a deficiency of either adenosine kinase (EC 2.7.1.20), adenine phosphoribosyltransferase (EC 2.4.2.7), or hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) abolishes the ability of adenine to cause cell death by interfering with pyrimidine synthesis;on the other hand, the pyrimidine starvation caused by adenosine is fully prevented only by a deficiency of adenosine kinase.Abbreviations WT wild-type line - AK adenosine kinase - APRT adenine phosphoribosyltransferase - HGPRT hypoxanthine guanine phosphoribosyltransferase - AD adenosine deaminase - A adenine - rA adenosine - I inosine - Hx hypoxanthine - dA 2-deoxyadenosine - dT 2-deoxythymidine - rU uridine - IMP inosine 5-monophosphate - AMP adenosine 5-monophosphate - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - PRPP phosphoribosylpyrophosphate - Amp aminopterin - TCA trichloracetic acid - ARA-A 9--darabinofuranosyladenine - Amp + dT medium normal (ERH) medium supplemented with Amp and dT     

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.     

Polyacrylamide gel electrophoresis of isoenzymes from Entamoeba species.

In this preliminary report, we describe a polyacrylamide gel electrophoresis technique for the resolution of isoenzyme patterns of four isolates of Entamoeba histolytica and one isolate of Entamoeba coli. Our findings were similar to previous findings for three enzyme systems: maleic enzyme (malate dehydrogenase [EC 1.1.1.40]), hexokinase (EC 2.7.1.1), and phosphoglucomutase (EC 2.7.5.1). We found preliminary evidence that glucosephosphate isomerase (EC 5.3.1.9) may also differentiate invasive amoebae from noninvasive amoebae, when the isoenzymes are separated by polyacrylamide gel electrophoresis, whereas this differentiation is not evident with starch-gel electrophoresis. We used an Rf system to relate isoenzyme band mobility to the migration distance of a standard E. histolytica strain (HK-9). The numerical identification of isoenzyme bands can simplify the grouping of isolates into zymodemes.     

Mechanisms of sulfadoxine resistance in Plasmodium falciparum

Three possible mechanisms of resistance to sulfadoxine were investigated in resistant Plasmodium falciparum: drug uptake, metabolism and alternate pathways. Uptake of [35S] sulfadoxine was markedly reduced in resistant plasmodia. By Thin Layer Radiochromatography it could be demonstrated that plasmodia do not metabolize sulfadoxine to pharmacologically inactive forms. Metabolism of sulfadoxine to the toxic analog of dihydropteroate is reduced in resistant plasmodia. Para-aminobenzoic acid (pABA) is not an essential nutrient for sulfonamide-resistant plasmodia. Instead, they seem to be able to synthesize pABA de novo. Four enzymes of the respective biosynthetic chain were demonstrated in isolated plasmodia: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase (EC 4.2.1.15), shikimate dehydrogenase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71) and pABA synthetase. We conclude that these three effects account for the reduced sulfonamide stress observed in the resistant parasite.     

Differences in substrate specificity and kinetic properties of the recombinant hexokinases HXK1 and HXK2 from Entamoeba histolytica

Entamoeba histolytica is responsible for amoebic colitis and liver abscess in humans. Entamoeba dispar is a closely related, morphologically indistinguishable nonpathogenic species. The hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns distinguish the pathogenic and nonpathogenic species. Both species possess two hexokinases with very similar molecular mass and different isoelectric points. In order to understand the role of the two different isoenzymes from E. histolytica, we purified the recombinant hexokinases HXK1 and HXK2 and examined substrate spectrum and kinetic properties. The two enzymes displayed similar temperature and pH optima, they were inhibited strongly by AMP and ADP, not by glucose 6-phosphate. Both enzymes phosphorylated glucose well and were unable to phosphorylate fructose or galactose. We also detected significant differences. HXK1 was more sensitive to inhibition by AMP and ADP. Mannose was phosphorylated well by HXK1, but at a much lower rate by HXK2. We attempted to expand the substrate spectrum of E. histolytica HXK1 by modifying its active site to become similar to the active site of the fructose phosphorylating yeast hexokinase PII. None of the nine mutants gained any fructokinase activity, but all of them retained at least some glucokinase and mannokinase activity. Mannokinase activity was decreased drastically by two single amino acid exchanges, both of which contributed significantly to this effect. The data indicate that a complex interaction of a number of amino acid residues is necessary for the ability to phosphorylate a given hexose.     

Distribution of components of the guanosine 3',5'-phosphate system in rat caudate-putamen

The cellular distribution of guanylate cyclase (EC 4.6.1.2), guanosine 3',5'-phosphate (cyclic GMP), cyclic GMP-dependent protein kinase (EC 2.7.1.38), and cyclic GMP phosphodiesterase (EC 3.1.4.17) have been examined in the rostral rat caudate-putamen complex. Immunofluorescent staining for guanylate cyclase, cyclic GMP, and cyclic GMP-dependent protein kinase in fresh frozen caudate-putamen tissues is analogous to the immunoperoxidase localization in perfusion-fixed striatal slices. Homologous immunoreactivity in the cytoplasm and processes of ovoid and rounded neurons, 15-20 microns in diameter can be seen for these three components of the cyclic GMP system. Immunoreactive neurons are uniformly distributed throughout the caudate-putamen complex of all experimental tissue examined. Occasional large neurons, greater than 25 microns in diameter, in the ventral region of the striatum show immunoreactivity. Enzyme histochemical determination of the activities of guanylate cyclase and cyclic GMP phosphodiesterase show the medium-sized neuronal population (15-20 microns) contain hydrolytic activity for these proteins. Large- to medium-sized capillaries demonstrate guanylate cyclase synthetic activity, but the endothelial cells do not exhibit immunohistochemical staining. This suggests that physiological activity of an enzyme cannot be completely discerned through application of immunohistochemical procedures. Additionally, enzymatically detected guanylate cyclase histochemical activity was not uniformly distributed throughout the striatal neuropil. Enzyme histochemical detection of cyclic GMP phosphodiesterase demonstrates homologous cellular staining to guanylate cyclase enzymatic reactivity. The activity of the phosphodiesterase hydrolytic enzyme could be detected evenly distributed throughout the neuropil within cells 15-20 microns in diameter, analogous in cytoarchitecture to immunohistochemically visualized guanylate cyclase, cyclic GMP, and protein kinase elements. Ultrastructural examination of rat caudate-putamen demonstrates that the immunoreactivity for the components of the cyclic GMP system is predominantly distributed within the medium-spiny neuron subtype of this structure. Occasional aspiny neurons demonstrate peroxidase immunoreactivity for the cyclase, cyclic GMP, and the protein kinase, as does the luminal surface of capillary endothelial cells. The subcellular distribution of the antigenic determinants for these three elements and the hydrolytic activity of the phosphodiesterase enzyme show proximity to one another and are confined to the postsynaptic region of asymmetrical, but not symmetrical, terminal boutons. The asymmetrical terminal population of the caudate-putamen is derived from striatal afferents from the neocortex, intralaminar thalamus, and substantia nigra, and to a lesser extent the intrinsic striatal circuitry.(ABSTRACT TRUNCATED AT 400 WORDS)     

GTP-dependent RNA 3'-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus

We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.