5′-Nucleotidase

Summary Determinations of pH activity curves and of Michaelis constants of 5-nucleotidases in organs of rat and mouse indicate the heterogenity of the enzyme in these tissues. Electrophoretic analyses of homogenates and cell component fractions reveal the presence of 5-nucleotidase isoenzymes in the investigated tissues. At the acid as well as at the neutral pH five isoenzymes were found. In addition three alkaline phosphatases were found in the rat; in the mouse four alkaline phosphatase isoenzymes could be demonstrated. The different combinations of 5-nucleotidase isoenzymes in the investigated tissues possibly indicate different functions of the isoenzymes. A discussion is given of the correlations between the electrophoretic results and the histochemical findings.     

5′-Nucleotidase

Summary The distribution of 5-nucleotidase in several tissues of rat and mouse has been investigated. The enzyme is greatly specific in dephosphorylating 5-nucleotides. Two 5-nucleotidases seem to exist: one showing greatest activity at pH 5.0, while the other is most active at pH 7.0–7.5. The localization of these two enzymes is not identical. At the acid pH the deoxyribonucleotides are dephosphorylated faster than the ribonucleotides, while at the neutral pH the ribonucleotides are hydrolysed more rapidly. In contrast to the non-specific phosphatases the nucleotidases can be stimulated by magnesium and manganese ions. The acid nucleotidase can be considered as a lysosomal enzyme. The neutral nucleotidase can be involved in transport processes in capillaries and sinusoids. Other localizations of the enzyme suggest a role of the enzyme in the catabolism of nucleic acids.     

5′-Nucleotidase from Yeast,Having Special Affinity for 5′-AMP

Three kinds of diketopiperazines which have retarditive activity for the growth of plant seedlings and plant roots at concentrations ranging from 1 : 2,500 to 1 : 100,000, were isolated from the neutral fraction by extracting the cultured broth of Rosellinia necatrix. These three diketopiperazines have been proved to be l-prolyl-l-leucine anhydride, l-prolyl-l-valine anhydride and l-prolyl-l-phenylalanine anhydride respectively, and the last one seems to be a new diketo-piperazine.

Furthermore, a crystalline wax having m.p. 52°C, a physiologically inactive substance, was also isolated from the same neutral fraction and presumed to be the saturated hydrocarbon of n-pentacosane C₂₅H₅₂.     

5′-Nucleotidase in spinal meningeal compartments in the rat

Summary The distribution of 5-nucleotidase (5-Nu) is reported in spinal meninges of the rat on the basis of an immunohistochemical and enzyme histochemical investigation. Strong immunoreactivity was found in the arachnoid membrane and in the sheaths of the spinal roots as well as in septa subdividing the roots. Also the superficial layer of the ligamentum denticulatum showed enzyme staining. No immunoreactivity could be detected in the pia mater or along the spinal nerve roots outside the subarachnoid space. Within the arachnoid mater the immunoreactivity was concentrated in the basal zone of the arachnoid membrane, thus appearing as a narrow fluorescent band near the border of the dura. An accentuation of immunoreactivity could be observed in areas where small dural blood vessels approach the subarachnoid space. It is well known that adenine nucleotides released from neural and glial cells of the central nervous system finally reach the cerebrospinal fluid. We presume that 5-Nu in the arachnoid membrane and spinal root sheaths is responsible for the conversion of adenine nucleotides into adenosine and that this conversion is associated with the reabsorption process of cerebrospinal fluid which most probably also takes place in spinal meninges. Adenosine, the product of 5-nucleotidase, could play a role in the reabsorption process by its vasodilatatory effect on dural and epidural vessels.     

5′-Nucleotidase in PC12 cells as revealed by immunocytochemistry

5-Nucleotidase hydrolyzes 5-mononucleotides to their nucleosides but is also thought to have a function in neuronal differentiation and synapse formation. The distribution of the enzyme, a glycosyl-phosphatidylinositol-anchored sialoglycoprotein, was investigated in PC12 cells using immunofluorescence microscopy. 5-Nucleotidase was located both in intracellular compartments and at the cell surface. There was no principal difference in the cellular distribution between undifferentiated cells and after neuritogenic differentiation by nerve growth factor. Intracellularly, 5-nucleotidase often revealed a sickle-shaped perinuclear distribution and a dotted pattern throughout the cytoplasm, including that of neurites and growth cones. The intracellular distribution was clearly different from that of the synaptic vesicle protein synaptophysin. However, the dotted fluorescence resembled that obtained after uptake of the endosomal marker acridine orange. 5-Nucleotidase was present on the entire cell surface including all neurites formed after differentiation. There was no increase in 5-nucleotidase fluorescence at synapse-like contacts between the tips of neurites and other PC12 cells. Surfacelocated 5-nucleotidase could no longer be detected after the application of glycosyl-phosphatidylinositol-specific phospholipase C to cultured cells. This treatment did not affect PC12 cell differentiation. Our results thus reveal 5-nucleotidase both at the surface and within organelles and suggest that PC12 cells may be used as a model system for the study of the physiological function of 5-nucleotidase in neural cells.     

Elektronenmikroskopischer Nachweis der 5′-Nucleotidase im Keimzentrum der menschlichen Tonsille

Zusammenfassung An den Keimzentren menschlicher glutaraldehydfixierter Tonsillen untersuchten wir elektronenmikroskopisch die 5-Nucleotidaseaktivität nach der Methode vonWachstein undMeisel. Der Prozentsatz an positiv reagierenden Keimzentrumszellen war insgesamt gering. Unter allen Zelltypen des Keimzentrums fanden sich solche mit deutlich positiver Reaktion. Am häufigsten war die Fermentaktivität an Lymphozyten nachzuweisen, weiterhin auch an Germinozyten und Germinoblasten. Plasmazellen als die am weitesten differenzierten Zellen des Keimzentrums reagierten nur selten positiv. Bei allen Zellen lagen die Reaktionsniederschläge der Plasmamembran teils innen, teils außen an, wobei positive Membrananteile mit fermentnegativen wechselten. Es ist daher anzunehmen, daß das Ferment im Bereich der Plasmamembran lokalisiert ist. Diese Lokalisation entspricht den von verschiedenen Autoren an Zellen anderer Organe elektronenmikroskopisch erhobenen Befunden. Im Gegensatz zu den Resultaten lichtmikroskopischer Untersuchungen konnte im Keimzentrum auch eine Aktivität der Adenosintriphosphatase an den Zellmembranen nachgewiesen werden. Nach dieser elektronenmikroskopischen Untersuchung stellt die 5-N-ase bei pH 7,2 kein Markierungsenzym für einen bestimmten Zelltyp des Keimzentrums dar. Es ist aber noch zu klären, welche zytologischen Fermentaktivitäten sich im sauren pH-Bereich darstellen.

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Electron microscopic study of 5-nucleotidase in the germinal centres of human tonsils

Summary Activity of 5-nucleotidase within germinal centres of human tonsils has been investigated with the electron microscope. Tonsils were fixed in glutaraldehyde and incubated according to the method ofWachstein andMeisel. The percentage of positively reacting germinal centre cells was all together small. All types of germinal center cells can show 5 nucleotidase — activity (lymphocytes, germinocytes, germinoblasts, rarely plasmacells). The reaction product was deposited on the outer or inner face of the plasma membrane. This finding is in accordance with the results of other authors obtained in cell-types from other tissues. In contrast to light microscopical observations, activity of adenosintriphosphatase was also demonstrated on the plasma-membranes. As all cell-types can give a positive reaction, the demonstration of 5-nucleotidase cannot be used for marking one specific cell-type within the germinal centres.

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Diese Untersuchung wurde mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft durchgeführt.     

5′-Nucleotidase activity in the pineal organ of the pike

Summary To date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear synaptic vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.     

5′-Nucleotidase Activity in Improved Inosine-producing Mutants of Bacillus subtilis

An inosine- and guanosine-producing strain, AJ11100, of Bacillus subtilis could not grow in the minimum medium supplemented with 50 µg of sulfaguanidine per ml. When sulfaguanidine resistant mutants were derived from AJ11100, the sulfaguanidine resistance was frequently accompanied by xanthine requirement. All the xanthine auxotrophic mutants required a large amount of xanthine for cell growth and inosine accumulation. Revertants were then derived from one of the xanthine auxotrophic mutants, AJ11101, and improved inosine producers were obtained. The best mutant, AJ11102, accumulated 20.6 g of inosine per liter.

Furthermore, enzyme activities of inosine 5′-monophosphate (IMP) dehydrogenase, 5′-nucleotidase and phosphoribosyl pyrophosphate (PRPP) amidotransferase were assayed to investigate why AJ11102 accumulated an increased amount of inosine. The results showed that the increase of specific activity of 5′-nucleotidase contributed much to the increased accumulation of inosine.     

Die saure 5′-Nucleotidase im Zentralnervensystem der wei\en Ratte

Zusammenfassung Bei ausgewachsenen Ratten beiderlei Geschlechts wurden die Bedingungen für den Nachweis der sauren 5-Nucleotidase (AMP) bestimmt. Die besten Ergebnisse wurden nach zweistündiger Immersionsfixation in Formol-Kalzium, mit anschließender Postfixation in derselben Lösung mit 0,88 M Saccharose erzielt. Die frei schwimmenden Schnitte wurden in folgender Lösung inkubiert (Modifikation der Methode von Wachstein und Meisel): 62,5 mg Adenosin-5-Phosphat (3,5 mM); 10 ml destilliertes Wasser; 5 ml 0,2 M Tris-Maleat-Puffer pH 7,55; 30 ml 0,2% Bleinitrat (3,6 mM) und 5 ml 0,1 M MgSO₄ (10 mM). Es wurde die Bedeutung der Substratmenge, des pH der Inkubationslösung und des Puffers sowie der Einfluß der Fixation auf die Enzymaktivität untersucht. Es ergaben sich drei Reaktionstypen, abhängig von den pH Optima (pH 3,5–pH 3,6; pH 4,5–pH 4,6 und pH 7,0–pH 7,1), die außer Verschiedenheiten im Charakter des Endproduktes auch unterschiedliches Verhalten in bezug auf die Dauer der Fixation zeigen. Bei der Verteilung der Aktivität bei pH 3,5–3,6 wird außer Golgi-Zonen und Lysosomen (ebenso wie bei pH 4,5–4,6) ein Reaktionsprodukt auch in den synaptischen Endigungen festgestellt. Bei pH 7,0 sind in erster Linie die Perikarya und die Fortsätze der Gliazellen in den verschiedenen Teilen des Endhirns positiv. Die stärkste Aktivität bei pH 3,5 bis 4,6 zeigen die Zellen des Vorderhorns und der Marginalzone des Rückenmarks, Nucleus n. hypoglossi des Rautenhirns, die Pyramidenzellen des Hippocampus und die Zellen der Granularschicht der Area dentata, Nucl. supraopticus und pars magnocellularis des Nucl. paraventricularis des End- und Zwischenhirns. — Die Ergebnisse sprechen für eine Heterogenität der AMP.

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Acid 5-nucleotidase in the central nervous system of white rats

Summary In adult white rats of both sexes the conditions for the best histochemical demonstration of the acid 5-nucleotidase (AMP) were determined. Best results were obtained with immersion fixation for 2 hours in formol calcium, followed by postfixation in the same solution, containing 0.88 M sucrose for 12–18 hours. The free floating sections were incubated in a solution representing a modification of the method of Wachstein and Meisel: 62.5 mg adenosine-5-phosphate (3.5 mM); 10 ml distilled water; 5 ml 0.2 M Tris-maleate buffer pH 7.55; 30 ml 0.2% lead nitrate and 5 ml 0.1 M magnesium sulfate. The method is suggested after investigation of the importance of the quantity substrate, pH of the medium, pH of the buffer and the influence of the fixation upon the enzyme activity. There are found 3 types of reaction at respective pH optimums: 3.5–3.6; 4.5–4.6 and 7.0–7.1, and beside differences in the character of the end product, they show also different behaviour corresponding to the time of fixation. In the distribution of the activity at pH 3.5–3.6 perhaps, beside the zones of Golgi and lysosomes (as it is at pH 4.5–4.6) reaction product is also found out in the synaptic endings. At pH 7.0 the picture is thoroughly different, especially the cell bodies and the processes of the glial cells are positive in the different parts of the brain. At pH 3.5–4.6 strongest activity show the cells of the anterior horn and the marginal zone of the spinal cord, Nucl. n. hypoglossi of the Medulla oblongata, the nuclei of the white matter and the cells of Purkinje in the cerebellum, the pyramidal cells of the Hippocampus, the cells of the granular layer of area dentata, nucl. supraopticus and pars magnocellularis of nucl. paraventricularis. The results support the opinion for heterogenity of AMP.

     

Elektronenmikroskopische Verteilung und Spezifität der sauren 5′-Nucleotidase im Zentralnervensystem der Ratte

Zusammenfassung Es wird eine Methode zur ultracytochemischen Darstellung der sauren 5-Nucleotidase im Zentralnervensystem der Ratte beschrieben. Die Ergebnisse werden mit der elektronenmikroskopischen Verteilung der sauren Phosphatase verglichen.In Rückenmark, Medulla oblongata und Endhirn sind beide Enzyme in den Cisternen und Bläschen des Golgi-Apparates sowie Lysosomen lokalisiert. Daneben kommen negative Golgi-Zonen und Lysosomen vor. Bei pH 5,5 tritt die saure Phosphatase zusätzlich in Kern und Ergastoplasma der Nervenzellen auf. Intralysosomal bestehen zwischen der sauren 5-Nucleotidase und Phosphatase Verteilungsdifferenzen.Gegenüber Hemmsubstanzen verhalten sich diese Enzyme unterschiedlich, wobei die Wirkung des Inhibitors von seiner Konzentration und dem untersuchten Areal abhängt.

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Electron microscopic distribution and specifity of acid 5-nucleotidase in the central nervous system of the rat

Summary A method for the ultracytochemical demonstration of acid 5-nucleotidase has been described in the central nervous system of the rat. The results were compared with those obtained for acid phosphatase.In the spinal cord, medulla oblongata and fore brain both enzymes are localized in the cisterns, vesicles and vacoules of the Golgi apparatus as well as in lysosomes. Some Golgi cisterns and lysosomes do not react. In addition at pH 5.5 acid phosphatase can be observed in the nucleus and in the ergastoplasm of the nerve cells. Concerning the intralysosomal distribution these enzymes exhibit some differences.Following pretreatment with various inhibitors differences occur between acid 5-nucleotidase and phosphatase depending also from the concentration of the substance used and the region investigated.

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Herrn Dr. R. Gossrau, Anatomisches Institut der Universität Würzburg/BRD, danken wir für die Hilfe bei der Manuskriptanfertigung.     

Oxidative Inactivation of Brain Ecto-5′-Nucleotidase by Thiols/Fe2+ System

5-Nucleotidase, responsible for the conversion of adenosine-5-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe²⁺. The glutathione-mediated inactivation of 5-nucleotidase was potentiated remarkably by Fe²⁺, but not Cu²⁺, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe²⁺-mediated inactivation. In comparison, the glutathione/Fe²⁺ system was much more effective than the ascorbate/Fe²⁺ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H₂O₂ in the action of glutathione/Fe²⁺ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe²⁺ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5-monophosphate as substrate exhibited a modest protection against the glutathione/Fe²⁺ action, a remarkable protection was expressed by divalent metal ions such as Zn²⁺ or Mn²⁺. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe²⁺ resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5-nucleotidase, were found to be more effective in potentiating the Fe²⁺- mediated inactivation. Further, kinetic analyses indicate that Fe²⁺ and thiols inhibit the 5-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5-nucleotidase from brain membrane is one of proteins susceptible to thiols/ Fe²⁺-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe²⁺ and thiols to the enzyme molecule.     

5′-Nucleotidase from bovine brain cortex: effect of solubilization on enzyme kinetics and modulation

The kinetic characteristics and the EDTA inhibition of microsomal 5′-nucleotidase from bovine brain cortex were studied and compared with the properties of the enzyme solubilized with Lubrol WX. The K_m value after enzyme solubilization was not significantly different from that of the membrane-bound enzyme. Likewise, di- and trinucleotides performed a similar competitive inhibition of the two forms of the enzyme. In contrast, divalent cations inhibited the intact microsomal enzyme activity at the same concentrations in which they increased the soluble-enzyme activity. The solubilization of microsomal 5′-nucleotidase did not change the progressive and irreversible character of the EDTA inhibition, but the mechanism of the irreversible inhibition was different. The addition of divalent metal cations did not affect the irreversibility of either inhibition, even though the effect on the residual activities was different. The Arrhenius plot of the 5′-nucleotidase activity in intact microsomal fraction exhibited a well-defined break at 31 ± 0.1°C, whereas that of the solubilized enzyme was a straight line. It is concluded then that microsomal 5′-nucleotidase from bovine brain cortex does not require the membrane environment to express its activity, although the influence of this lipidic environment was evident in the differences observed in the enzyme activity modulation by EDTA, cations and temperature.     

Streptococcal 5′-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5′-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a K_m of 169 μm and a V_max of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5′-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg²⁺, Ca²⁺, or Mn²⁺. However, Zn²⁺ inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5′-nucleotidase activity and immune evasion properties.     

The 5′-Nor Aristeromycin Analogues of 5′-Deoxy-5′-Methylthioadenosine and 5′-Deoxy-5′-Thiophenyladenosine

To extend the potential of 5′-noraristeromycin (and its enantiomer) as potential antiviral candidates, the enantiomers of the carbocyclic 5′-nor derivatives of 5′-methylthio-5′-deoxyadenosine and 5′-phenylthio-5′-deoxyadenosine have been synthesized and evaluated. None of the compounds showed meaningful antiviral activity.     

Piracetam Prevents Scopolamine-Induced Memory Impairment and Decrease of NTPDase, 5′-Nucleotidase and Adenosine Deaminase Activities

Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5′-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5′-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5′-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5′-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5′-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.     

5′-Nucleotidase and adenosine deaminase in developing fetal guinea pig brain and the effect of maternal hypoxia

The activity of key enzymes of adenosine metabolism was studied in the developing fetal guinea pig brain. The activities of 5-nucleotidase and adenosine deaminase were determined in the brains of fetal guinea pigs at 30, 35, 40, 45, 50, 55, and 60 days of gestation. The level of 5-nucleotidase activity was extremely low at 30 and 35 days of gestation but increased rapidly during the 40 to 60 day period. The enzyme activity increased in the presence of Mg²⁺ with the Mg²⁺-dependent activation increasing with the age of gestation. This Mg²⁺-dependent activity was primarily associated with the membrane fraction. Prenatal hypoxia significantly increased the fetal brain M²⁺-independent 5-nucleotidase activity at 45 days of gestational age and beyond. Prior to this age, no effect was evident. Furthermore, following hypoxia, the Mg²⁺-dependent activation of 5-nucleotidase activity was lost. The activity of adenosine deaminase was present at 30 days of gestation and, unlike 5-nucleotidase, it remained at the same level until 60 days. The results indicate that the term fetal guinea pig brain has the enzymatic mechanisms of adenosine metabolism and thus the potential for adenosine-mediated regulation of cerebrovasculature during hypoxia.     

Substrate Properties of Adenosine- and Uridine- 3′- Phenylphosphonates for 3′-Nucleotidase/Nucleases

Abstract

Adenosine- and uridine- 3′-phenylphosphonates have been synthesized and evaluated as substrates of 3′-nucleotidase/nucleases. No other nucleases hydrolyzed these compounds. Since V_max values for the adenosine derivative were comparable to those for 3 -AMP and the apparent K_m were 1.4–2.6 mM, it may be a useful substrate.

     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.