Publications

• Purification and properties of human placental ATP diphosphohydrolase.

  Christoforidis S., Papamarcaki T., Galaris D., Kellner R., Tsolas O.

  ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purificati ... >> More

  ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5'-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8-azido-[alpha-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzymic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting. << Less

  Eur. J. Biochem. 234:66-74(1995) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles.

  Lopez-Avalos M.D., Uccelletti D., Abeijon C., Hirschberg C.B.

  In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significa ... >> More

  In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes. << Less

  Glycobiology 11:413-422(2001) [PubMed] [EuropePMC]

• Characterization of gdp1+ as encoding a GDPase in the fission yeast Schizosaccharomyces pombe.

  Sanchez R., Franco A., Gacto M., Notario V., Cansado J.

  We have isolated the gdp1+ gene from Schizosaccharomyces pombe coding for a membrane protein with guanosine diphosphatase (GDPase) activity, which is highly homologous to Golgi GDPases isolated from other yeast species. The gdp1+ product, Gdp1p, displays both GDPase and uridine diphosphatase (UDPa ... >> More

  We have isolated the gdp1+ gene from Schizosaccharomyces pombe coding for a membrane protein with guanosine diphosphatase (GDPase) activity, which is highly homologous to Golgi GDPases isolated from other yeast species. The gdp1+ product, Gdp1p, displays both GDPase and uridine diphosphatase (UDPase) activities in vitro, with a strong dependence for calcium and manganese cations. The observation of a defect in N-glycosylation of invertase in S. pombe Deltagdp1 cells together with the ability of gdp1+ to functionally complement the defective O-mannosylation of chitinase in Saccharomyces cerevisiae cells disrupted in the GDA1 gene (gdp1+ homolog), suggests a main role for Gdp1p in protein glycosylation in fission yeast. << Less

  FEMS Microbiol. Lett. 228:33-38(2003) [PubMed] [EuropePMC]

• Purification and characterization of a guanosine diphosphatase activity from calf liver microsomal salt wash proteins.

  Raychaudhuri P., Ghosh S., Maitra U.

  A potent guanosine diphosphatase activity that hydrolyzes GDP to 5'-GMP + Pi has been isolated and purified from the salt wash proteins of calf liver microsomes. The purified enzyme, a monomeric protein of approximate Mr 46,000, possesses nucleotide substrate specificity since, among the nucleosid ... >> More

  A potent guanosine diphosphatase activity that hydrolyzes GDP to 5'-GMP + Pi has been isolated and purified from the salt wash proteins of calf liver microsomes. The purified enzyme, a monomeric protein of approximate Mr 46,000, possesses nucleotide substrate specificity since, among the nucleoside diphosphates and triphosphates tested, only GDP and UDP are hydrolyzed by the enzyme. The relative affinity of the enzyme for GDP is, however, much higher than for UDP. The effect of the enzyme on the binary complex formed between eukaryotic initiation factor 2 (eIF-2) and GDP has also been investigated. The enzyme neither hydrolyzes GDP bound to eIF-2 nor catalyzes the exchange of eIF-2-bound GDP with GTP even in the presence of Met-tRNAf. The enzyme, therefore, is presumably not involved in recycling of eIF-2 in eukaryotic polypeptide chain initiation reaction. The possible biological function of the enzyme in maintaining the cellular pool of GTP-GDP is discussed. << Less

  J Biol Chem 260:8306-8311(1985) [PubMed] [EuropePMC]

• ire-1-dependent transcriptional up-regulation of a lumenal uridine diphosphatase from Caenorhabditis elegans.

  Uccelletti D., O'Callaghan C., Berninsone P., Zemtseva I., Abeijon C., Hirschberg C.B.

  Lumenal ecto-nucleoside tri- and di-phosphohydrolases (ENTPDases) of the secretory pathway of eukaryotes hydrolyze nucleoside diphosphates resulting from glycosyltransferase-mediated reactions, yielding nucleoside monophosphates. The latter are weaker inhibitors of glycosyltransferases than the fo ... >> More

  Lumenal ecto-nucleoside tri- and di-phosphohydrolases (ENTPDases) of the secretory pathway of eukaryotes hydrolyze nucleoside diphosphates resulting from glycosyltransferase-mediated reactions, yielding nucleoside monophosphates. The latter are weaker inhibitors of glycosyltransferases than the former and are also antiporters for the transport of nucleotide sugars from the cytosol to the endoplasmic reticulum (ER) and Golgi apparatus (GA) lumen. Here we describe the presence of two cation-dependent nucleotide phosphohydrolase activities in membranes of Caenorhabditis elegans: one, UDA-1, is a UDP/GDPase encoded by the gene uda-1, whereas the other is an apyrase encoded by the gene ntp-1. UDA-1 shares significant amino acid sequence similarity to yeast GA Gda1p and mammalian UDP/GDPases and has a lumenal active site in vesicles displaying an intermediate density between those of the ER and GA when expressed in S. cerevisiae. NTP-1 expressed in COS-7 cells appeared to localize to the GA. The transcript of uda-1 but not those of two other C. elegans ENTPDase mRNAs (ntp-1 and mig-23) was induced up to 3.5-fold by high temperature, tunicamycin, and ethanol. The same effectors triggered the unfolded protein response as shown by the induction of expression of green fluorescent protein under the control of the BiP chaperone promoter and the UDP-glucose:glycoprotein glucosyltransferase. Up-regulation of uda-1 did not occur in ire-1-deficient mutants, demonstrating the role of this ER stress sensor in this event. We hypothesize that up-regulation of uda-1 favors hydrolysis of the glucosyltransferase inhibitory product UDP to UMP, and that the latter product then exits the lumen of the ER or pre-GA compartment in a coupled exchange with the entry of UDP-glucose, thereby further relieving ER stress by favoring protein re-glycosylation. << Less

  J. Biol. Chem. 279:27390-27398(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• CD39L2, a gene encoding a human nucleoside diphosphatase, predominantly expressed in the heart.

  Yeung G., Mulero J.J., McGowan D.W., Bajwa S.S., Ford J.E.

  E-NTPDases are extracellular enzymes that hydrolyze nucleotides. The human E-NTPDase gene family currently consists of five reported members (CD39, CD39L1, CD39L2, CD39L3, and CD39L4). Both membrane-bound and secreted family members have been predicted by encoded transmembrane and leader peptide m ... >> More

  E-NTPDases are extracellular enzymes that hydrolyze nucleotides. The human E-NTPDase gene family currently consists of five reported members (CD39, CD39L1, CD39L2, CD39L3, and CD39L4). Both membrane-bound and secreted family members have been predicted by encoded transmembrane and leader peptide motifs. In this report, we demonstrate that the human CD39L2 gene is expressed predominantly in the heart. In situ hybridization results from heart indicate that the CD39L2 message is expressed in muscle and capillary endothelial cells. We also show that the CD39L2 gene encodes an extracellular E-NTPDase. Flow cytometric experiments show that transiently expressed CD39L2 is present on the surface of COS-7 cells. Transfected cells also produce recombinant glycosylated protein in the medium, and this process can be blocked by brefeldin A, an inhibitor of the mammalian secretory pathway. The enzymology of CD39L2 shows characteristic features of a typical E-NTPDase, but with a much higher degree of specificity for NDPs over NTPs as enzymatic substrates. The kinetics of the ADPase activity exhibit positive cooperativity. The predominance of CD39L2 expression in the heart supports a functional role in regulating platelet activation and recruitment in this organ. << Less

  Biochemistry 39:12916-12923(2000) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• The ER UDPase ENTPD5 promotes protein N-glycosylation, the Warburg effect, and proliferation in the PTEN pathway.

  Fang M., Shen Z., Huang S., Zhao L., Chen S., Mak T.W., Wang X.

  PI3K and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinases that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. We report here that ENTPD5, an endoplasmic reticulum (ER) e ... >> More

  PI3K and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinases that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. We report here that ENTPD5, an endoplasmic reticulum (ER) enzyme, is upregulated in cell lines and primary human tumor samples with active AKT. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN null cells causes ER stress and loss of growth factor receptors. ENTPD5, together with cytidine monophosphate kinase-1 and adenylate kinase-1, constitute an ATP hydrolysis cycle that converts ATP to AMP, resulting in a compensatory increase in aerobic glycolysis known as the Warburg effect. The growth of PTEN null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an integral part of the PI3K/PTEN regulatory loop and a potential target for anticancer therapy. << Less

  Cell 143:711-724(2010) [PubMed] [EuropePMC]