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PubMed1999

Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate.

Discovery of a structurally conserved metal-dependent lithium-inhibited phosphomonoesterase protein family has identified several potential cellular targets of lithium as used to treat manic depression. Here we describe identification of a novel family member using a "computer cloning" strategy. Human and murine cDNA clones encoded proteins sharing 92% identity and were highly expressed in kidney. Native and recombinant protein harbored intrinsic magnesium-dependent bisphosphate nucleotidase activity (BPntase), which removed the 3'-phosphate from 3'-5' bisphosphate nucleosides and 3'-phosphoadenosine 5'-phosphosulfate with Km and Vmax values of 0.5 microM and 40 micromol/min/mg. Lithium uncompetitively inhibited activity with a Ki of 157 microM. Interestingly, BPntase was competitively inhibited by inositol 1,4-bisphosphate with a Ki of 15 microM. Expression of mammalian BPntase complemented defects in hal2/met22 mutant yeast. These data suggest that BPntase's physiologic role in nucleotide metabolism may be regulated by inositol signaling pathways. The presence of high levels of BPntase in the kidney are provocative in light of the roles of bisphosphorylated nucleotides in regulating salt tolerance, sulfur assimilation, detoxification, and lithium toxicity. We propose that inhibition of human BPntase may account for lithium-induced nephrotoxicity, which may be overcome by supplementation of current therapeutic regimes with inhibitors of nucleotide biosynthesis, such as methionine.

Amino Acid Sequence↗

PubMed1999

Tissue-specific regulation of the ecto-5'-nucleotidase promoter. Role of the camp response element site in mediating repression by the upstream regulatory region.

We have isolated the 5' region of the ecto-5'-nucleotidase (low K(m) 5'-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5'-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5' of the ecto-5'-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.

5'-Nucleotidase↗

PubMed2006

Structure of pyrimidine 5'-nucleotidase type 1. Insight into mechanism of action and inhibition during lead poisoning.

Eukaryotic pyrimidine 5'-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5'-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35 A resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25 A, 2) phosphoenzyme intermediate analog at 2.30 A, 3) product-transition complex analog at 2.35 A, and 4) product complex at 2.1A resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35 A) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.

5'-Nucleotidase↗

PubMed2005

5'-nucleotidase activity is altered by hypo- and hyperthyroidism in platelets from adult rats.

Thyroid disorders are associated to a number of vascular diseases that involve processes such as platelet aggregation and vascular tone control. Since, these processes can be also affected by ATP, ADP and adenosine levels, we investigate the hydrolysis of these nucleotides in platelets from hyperthyroid, hypothyroid, and hypothyroid with hormonal replacement rats. Hyperthyroidism was induced by daily injections of L-thyroxine (T4) 25 microg/100 g body weight for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) for 14 days. In the hormonal replacement group, hypothyroid rats were injected with T4 (5 microg/100 g body weight, i.p.) for 5 days. The AMP hydrolysis by platelets was increased 49% in hyperthyroid rats and decreased 50% in response to hypothyroidism, while the ATP and ADP hydrolysis was not altered in both groups. Besides, the T4 replacement significantly reversed the inhibition of the AMP hydrolysis observed in hypothyroid rats. Our findings indicate that the thyroid disorders affect the 5'-nucleotidase activity and consequently can alter the adenosine levels in a reversible manner in platelet fraction. Since, adenosine is able to inhibit platelet aggregation and acts as a potent vasodilator, these results can contribute to a better comprehension of the vascular events described in thyroid disorders.

5'-Nucleotidase↗

PubMed2003

Role of IMP-selective 5'-nucleotidase (cN-II) in hematological malignancies.

Cytotoxic nucleoside analogs (NA) are important in the treatment of hematologic malignancies. The NA in routine clinical use include the pyrimidine analog cytosine arabinoside (ara-c), which is extensively used in the treatment of acute leukemias, and the purine analogs, cladribine and fludarabine. These drugs have mostly been used in the treatment of low grade hematological malignancies. NA become therapeutically effective only after phosporylation to the triphosphate level. The 5'-nucleotidases (5'-NTs) dephosphorylate the monophosphate form of NA and, therefore, may affect the pharmacological activity of these antimetabolites in the clinic. Several 5'-NTs attached to membranes or present in the cytosol or in mitochondria are present in mammalian cells. cN-II, an IMP-selective 5'-NT, participates in the regulation of purine deoxyribonucleotide metabolism. cN-II opposes the action of the salvage enzymes by dephosphorylating purine nucleoside mononphosphates to purine nucleosides. Due to its phosphotransferase activity, cN-II can also phosphorylate inosine and 2',3'-dideoxyribonucleosides utilizing IMP as a phosphate donor. The observation that cytosolic cN-II is able to phosphorylate purine nucleosides has initiated studies on its potential participation in the metabolism of anticancer agents and in the development of cN-II inhibitory substances. In this review, we highlight the current knowledge concerning cN-II activity and regulation of intracellular deoxyribonucleotide pools and it role in hematological malignancies.

5'-Nucleotidase↗

PubMed2004

Identification of the 5'-nucleotidase activity altered in neurological syndromes.

5'-Nucleotidases comprise a family of enzymes involved in the regulation of intracellular and extracellular nucleotide concentration. There is increasing knowledge about an involvement of these activities in the aetiology of neurological disorders. In this paper we present a protocol for the identification of the altered enzyme in fibroblasts primary culture from patients and controls.

5'-Nucleotidase↗

PubMed2004

The genetic basis of the interaction between pyrimidine 5' nucleotidase I deficiency and hemoglobin E.

We have previously described a family in which the interaction between pyrimidine 5' nucleotidase I (P5N-I) deficiency and hemoglobin E resulted in severe haemolytic anaemia. In this study we explored the genetic basis of the severe clinical phenotype and look for evidence of the interaction between these conditions. A P5N-I gene mutation (IVS8 + 1-2delGT) was found in the family, confirming that the severe phenotype results from the interaction between two genetic diseases.

5'-Nucleotidase↗

PubMed2005

Exposure to human blood inactivates swine endothelial ecto-5'-nucleotidase.

Ecto-5'-nucleotidase (E5'N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5'N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5'N activity to 62% and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5'N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5'N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.

5'-Nucleotidase↗

PubMed2005

Expression of human ecto 5' nucleotidase in pig endothelial cells and its implication for adenosine production and xenotransplantation.

Human endothelial activity of ecto-5'-nucleotidase (E5'N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and antiaggregatory adenosine. We have shown in this study that human E5'N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5'N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.

5'-Nucleotidase↗

PubMed1992

5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route.

In hepatocytes, all newly synthesized plasma membrane (PM) proteins so far studied arrive first at the basolateral domain; apically destined proteins are subsequently endocytosed and sorted to the apical domain via transcytosis. A mechanism for the sorting of newly synthesized glycophosphatidylinositol (GPI)-linked proteins has been proposed whereby they associate in lipid microdomains in the trans-Golgi network and then arrive at the apical domain directly. Such a mechanism poses a potential exception to the hepatocyte rule. We have used pulse-chase techniques in conjunction with subcellular fractionation to compare the trafficking of 5' nucleotidase (5NT), an endogenous GPI-anchored protein of hepatocytes, with two transmembrane proteins. Using a one-step fractionation technique to separate a highly enriched fraction of Golgi-derived membranes from ER and PM, we find that both 5NT and the polymeric IgA receptor (pIgAR) traverse the ER and Golgi apparatus with high efficiency. Using a method that resolves PM vesicles derived from the apical and basolateral domains, we find that 5NT first appears at the basolateral domain as early as 30 min of chase. However the subsequent redistribution to the apical domain requires > 3.5 h of chase to reach steady state. This rate of transcytosis is much slower than that observed for dipeptidylpeptidase IV, an apical protein anchored via a single transmembrane domain. We propose that the slow rate of transcytosis is related to the fact that GPI-linked proteins are excluded from clathrin-coated pits/vesicles, and instead must be endocytosed via a slower nonclathrin pathway.

5'-Nucleotidase↗

PubMed1979

Effects of cytoskeletal perturbant drugs on ecto 5'-nucleotidase, a concanavalin A receptor.

Differences in cell morphology, concanavalin A-induced receptor redistributions, and the cooperativity of the inhibition of 5'-nucleotidase (AMPase) by concanavalin A (Con A) have been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma cells treated with microfilament- and microtubule-perturbing drugs. By scanning electron microscopy MAT-C1 cells exhibit a highly irregular surface, covered with microvilli extending as branched structures from the cell body. MAT-A, MAT-B, and MAT-B1 cells have a more normal appearance, with unbranched microvilli, ruffles, ridges, and blebs associated closely with the cell body. MAT-C cells have an intermediate morphology. Treatment of MAT-A, MAT-B, or MAT-B1 cells with Con A causes rapid redistribution of Con A receptors. Both cytochalasins and colchicine cause alternations in the receptor redistributions. Receptors on MAT-C1 cells are highly resistant to redistribution, even in the presence of cytoskeletal perturbant drugs. The cooperativity of the inhibition of AMPase by Con A was investigated in MAT-A and MAT-C1 cells. Untreated cells exhibit no cooperativity. If either subline is treated with colchicine, cytochalasin B or D, or dibucaine, cooperativity is observed. Lumicolchicine has no effect. Theophylline or dibutyryl cyclic AMP prevents the effects of either colchicine or cytochalasin. The concentration required for half-maximal induction of cooperativity is 0.3--0.4 microM for both colchicine and cytochalasin D, which is in the appropriate range for specific microtubule and microfilament disruptions. The effectiveness of the cytochalasins (E greater than D greater than B) is consistent with their known effects on microfilaments. No direct correlation was observed between the induction of cooperativity and drug-induced changes in Con A receptor redistribution or cell morphology. The morphology of MAT-A cells is grossly altered by cytochalasins or dibucaine and somewhat less by colchicine. MAT-C1 cells exhibit more minor alterations in morphology as a result of these drug treatments. The results of this study indicate that the inhibition of AMPase, which is a Con A receptor, is a different process from the redistribution of the bulk of the Con A receptors, possibly short range membrane interactions rather than global effects on the cell.

Animals↗

PubMed1976

5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory populations.

Mouse resident peritoneal macrophages display sufficient 5'-nucleotidase activity to hydrolyze 58 nm AMP/min per cell protein. This activity increases approximately 163 nm AMP/min per mg after 72 h in culture. The enzyme is renewed in unstimulated cells with a half-time of 13.9 h. The activity is not reduced by treatment of intact cells with a variety of proteolytic enzymes, including trypsin, pronase, urokinase, and plasmin. Cells obtained from an inflammatory exudate have diminished or absent levels of enzyme activity. Endotoxin-elicited cells display enzyme activitiy of 20.9 nm AMP/min per mg, while thioglycollate-stimulated macrophages have no detectable activity. The reduced level of activity in endotoxin-stimulated cells is due to their elevated rate of enzyme degradation, with a half-time of 6.9 h. Their rate of enzyme synthesis is essentially normal. No evidence for latent enzyme activity could be obtained in thioglycollate-stimulated cells, nor do these cells produce any inhibition of normal cell enzyme activity. Serum deprivation reduces the enzyme activity of resident cells to about 45% of control activity. These conditions do not significantly affect the rate of enzyme synthesis, but again are explainable by an increase in the rate of enzyme degradation. Pinocytic rate is elevated in endotoxin-stimulated cells which show a more rapid rate of enzyme degradation than unstimulated cells do. However, in serum-free conditions, the rate of enzyme degradation is doubled with no change in the pinocytic rate of the cells.

Animals↗

PubMed2001

Expression of pyrimidine 5'-nucleotidase subclass I during erythrocyte maturation in rats.

The subclass I enzyme of rat pyrimidine 5'-nucleotidase (P5N-I), which preferentially hydrolyzes (deoxy)CMP and UMP, is distributed specifically in red blood cells (RBCs), and its activity increases approximately six-fold as compared to the control value after erythropoietic induction by phenylhydrazine administration. In this study, we detected rat P5N-I protein by using antibodies against the chicken P5N-I enzyme. The molecular mass of rat P5N-I was approximately 37 kDa, as estimated by gel filtration chromatography and Western blot analysis. The pI value of the enzyme was approximately 5.7. This protein band was detected only in RBC lysate extract, i.e., not in cytosol from the erythropoietic spleen. Protein mass of the P5N-I enzyme, estimated by immunoblot analysis, was increased in proportion to the enzyme activity after erythropoietic induction in rats. No phosphorylation of the enzyme protein was detected by immunoblot analysis with anti-phosphoserine or anti-phosphotyrosine antibody. In conclusion, these findings indicate that the rat P5N-I enzyme is expressed specifically in reticulocytes and may therefore be essential in the maturation process of rat erythrocytes.

5'-Nucleotidase↗

PubMed2000

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium: an attempt to reactivate the 5'-nucleotidase activity with Mg++ ion supply.

Enzyme-histochemical demonstration of lymphatic vessels in the golden hamster periodontium was performed on cryostat sections using the 5'-nucleotidase (5'-Nase) staining method by light microscopy and backscattered electron imaging of scanning electron microscopy. The inhibition of the 5'-Nase activity by decalcification was cancelled by the Mg++ ion supply. The reaction products of 5'-Nase activity were produced on the lymphatic endothelial cells and the tubular structures of lymphatic vessels were seen as highlights by backscattered electron imaging. The invasion of 5'-Nase-positive lymphatic vessels into the alveolar bone from the periodontium was found in the present study.

5'-Nucleotidase↗

PubMed1999

5'-Nucleotidase as a marker of both general and local inflammation in rheumatoid arthritis patients.

OBJECTIVES: To evaluate measurements of serum and synovial fluid 5'-nucleotidase (5'N) activity as a marker of general and local inflammation in arthritis, and to resolve a contradiction in the literature as to whether or not the activity of 5'N in the synovial fluids of rheumatoid arthritis (RA) patients is raised in comparison with that in the synovial fluids of other arthritis patients. METHODS: Assays for 5'N were carried out in the presence of inhibitors of other phosphatases, AMP deaminase and of 5'N itself. RESULTS: The 5'N activity in the synovial fluid of RA patients was both significantly higher (mean 1.7-fold) and had a greater variance than that in the synovial fluids of other arthritis patients, and the contradiction in the literature was resolved. There was a strong correlation between the 5'N activity in the sera of RA patients and their erythrocyte sedimentation rate. There was no significant correlation between the 5'N in the serum and synovial fluid for the RA patients, in marked contrast to the strong correlation between the two 5'N activities shown by the osteoarthritis patients. The 5'N activity was greater in the synovial fluid than in the serum for virtually all the patients, showing that it was being made locally. CONCLUSIONS: The 5'N activity in the serum (which came mostly from the liver) could be used as a marker of general inflammation, whereas the 5'N in the synovial fluid was mostly produced locally, and could be used as a marker of joint inflammation, particularly for the RA patients.

5'-Nucleotidase↗

PubMed1998

Expression and enzymic activity of ecto 5'-nucleotidase in the human male genital tract.

Human 5'-nucleotidase (5'-NT, EC 3.1.3.5) is an enzyme that hydrolyzes nucleotides such as AMP or IMP (inosine 5'-monophosphate) into inorganic phosphate and the respective nucleoside. It has been suggested that the enzyme acts as a scavenger of injured cell or membrane components or as a supplier of adenosine. We have purified to homogeneity human 5'-NT, a 69-kDa glycoprotein containing a glycosylphosphatidylinositol anchor, present in human seminal fluid. With use of a polyclonal rabbit antiserum against the protein, a strong immunoreaction was detected in prostatic epithelium, exceeding that in placental syncytiotrophoblast and amnion cells. A slightly less intense immunoreaction was present in some cells of seminal vesicle epithelium and in vesicular intraluminal secretion. In the epididymis, only the apical cell portion and particularly the stereocilia of the epididymal principal cells, as well as clusters of small nonciliated cells in the efferent ductules, were immunoreactive. In the testis, no immunoreactive cells at all were detected, and likewise no clear-cut signal was observed in testicular and epididymal spermatozoa. The immunohistochemical results were coincident with Western blots prepared from homogenates of the respective tissues. Reverse transcription-polymerase chain reaction studies were performed with primers derived from the sequence of human placental ecto 5'-NT. Using human placenta as a reference tissue, positive results were obtained in the epididymis, seminal vesicle, and prostate, but not in the testis. On Northern blots, we determined the size of the mRNA at 2.4 kilobases. The relatively strong expression of 5'-NT in the human male accessory sex glands points to a potential regulatory role of the enzyme during posttesticular modification of the sperm surface.

5'-Nucleotidase↗

PubMed2000

Respective value of alkaline phosphatase, gamma-glutamyl transpeptidase and 5' nucleotidase serum activity in the diagnosis of cholestasis: a prospective study of 80 patients.

We studied the value of alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), and 5'-nucleotidase (5'-NU) activities in the diagnosis of intrahepatic (IHC) versus extra-hepatic cholestasis (EHC). Eighty patients were included prospectively. All presented with cholestasis as defined by a concomitant increase in at least two of three cholestatic enzymes (AP, GGT, 5'-NU), a low cytolytic ratio (alanine aminotransferase/AP [xN/xN] < or = 5), and no evidence for associated liver tumor. We compared 43 patients with IHC due to chronic liver disease to 37 patients with EHC due to main bile duct obstruction. Fasting blood samples for activity determination (AP, GGT, 5'-NU) were taken before performing liver biopsy in cases of IHC and before endoscopic or surgical management in cases of EHC. Enzyme activities were compared using univariate and multivariate analysis. AP (276 IU/L [35-3,140] vs. 123 IU/L [37-699]: p < 0.0001), GGT (595 IU/L [98-5,200] vs. 211 IU/L [38-925]; p < 0.0001), and 5'-NU (32 IU/L [10-142] vs. 16 IU/L [4-107]: p < 0.0003) were significantly higher in EHC when compared to IHC. Only in GGT and 5'-NU activities were independent variables significantly linked to the mechanism of cholestasis. In IHC, the ratio GGT/5'-NU (xN/xN) was significantly lower than in EHC (2.8 [0.7-7.2] vs. 3.7 [1.8-10.5]: p < 0.006). A threshold of GGT/5'-NU < 1.9 had a sensitivity of 40% and a specificity of 100% for the diagnosis of IHC. Although such hepatobiliary enzymes cannot be regarded as diagnostic, they can provide useful information to orientate the clinician in the diagnosis of cholestasis.

Adolescent↗

PubMed1996

The SAL1 gene of Arabidopsis, encoding an enzyme with 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast.

A cDNA library in a yeast expression vector was prepared from roots of Arabidopsis exposed to salt and was used to select Li(+)-tolerant yeast transformants. The cDNA SAL1 isolated from one of these transformants encodes a polypeptide of 353 amino acid residues. This protein is homologous to the HAL2 and CysQ phosphatases of yeast and Escherichia coli, respectively. Partial cDNA sequences in the data bases indicate that rice produces a phosphatase highly homologous to SAL1 and that a second gene homologous to SAL1 exists in Arabidopsis. The SAL1 protein expressed in E. coli showed 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. In yeast, SAL1 restored the ability of a hal2/met22 mutant to grow on sulfate as a sole sulfur source, increased the intracellular Li+ tolerance, and modified Na+ and Li+ effluxes. We propose that the product of SAL1 participates in the sulfur assimilation pathway as well as in the phosphoinositide signaling pathway and that changes in the latter may affect Na+ and Li+ fluxes.

Amino Acid Sequence↗