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Terence L. Kirley - One of the best experts on this subject based on the ideXlab platform.
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mutagenesis of two conserved tryptophan residues of the e type atpases inactivation and conversion of an ecto Apyrase to an ecto ntpase
Biochemistry, 1999Co-Authors: Thomas M. Smith, Stephanie Ann Lewis Carl, Terence L. KirleyAbstract:A human brain E-type ATPase (HB6 ecto-Apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-Apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-Apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-Apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-Apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-Apyrase to an ecto-NTPase.
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glycosylation is essential for functional expression of a human brain ecto Apyrase
Biochemistry, 1999Co-Authors: Thomas M. Smith, Terence L. KirleyAbstract:The importance of N-linked glycosylation for the function and oligomerization of an E-type ATPase was examined by using tunicamycin and peptide N-glycosidase F, two agents used to prevent and remove glycosylations, respectively. The cDNA encoding a human ecto-Apyrase (HB6), predicted to have seven N-linked glycosylation sites, was transiently expressed in mammalian COS cells and the resulting membrane preparations were treated with peptide N-glycosidase F (PNGase-F). PNGase-F caused a decrease in the apparent molecular weight of the protein (consistent with glycan removal) and a decrease in enzymatic activity over time. The ecto-Apyrase was also expressed in the presence of tunicamycin, which completely prevented N-linked glycosylation, resulting in a nonglycosylated core protein devoid of ATP and ADP hydrolyzing activity. However, control and tunicamycin-treated cells expressed the enzyme to similar levels and localization. Interestingly, the quaternary structure of this E-type ATPase appears to be dependent upon the presence of glycan chains. The glycosylated ecto-Apyrase exists as a homodimer in situ as assessed by both size-exclusion chromatography of detergent-solubilized ecto-Apyrase and cross-linking of membrane-bound ecto-Apyrase, in contrast to the enzymatically deglycosylated ecto-Apyrase and the tunicamycin-treated ecto-Apyrase. These results suggest that glycosylation is necessary for homooligomerization and nucleotide hydrolyzing activity, but not for expression and plasma membrane localization of the E-type ATPase. Similar results were obtained with another human ecto-Apyrase, CD39, suggesting that the importance of glycosylation may be generalized to all membrane-bound E-type ATPases.
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Cloning, sequencing, and expression of a human brain ecto-Apyrase related to both the ecto-ATPases and CD39 ecto-Apyrases
Biochimica et Biophysica Acta, 1998Co-Authors: Thomas M. Smith, Terence L. KirleyAbstract:Abstract An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-Apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-Apyrase. However, this hydrolysis ratio is intermediate between that observed for the ecto-ATPases and the CD39 ecto-Apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141–214). Quantitative analyses of amino acid identities and similarities between this ecto-Apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both ecto-ATPases and CD39 ecto-Apyrases are derived.
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immunolocalization of the ecto atpase and ecto Apyrase in chicken gizzard and stomach purification and n terminal sequence of the stomach ecto Apyrase
Journal of Biological Chemistry, 1997Co-Authors: Stephanie Lewiscarl, Terence L. KirleyAbstract:We have examined the in vivolocalization of extracellular ecto-ATPase and ecto-Apyrase (ATPDase) in adult chicken gizzard and stomach by immunofluorescence and laser scanning confocal microscopy. In chicken gizzard, the ecto-ATPase was distributed in discrete clusters restricted to the sarcolemma of the smooth muscle cells. Anti-ecto-Apyrase antibody detected a single 80-kDa band (putative Apyrase) in Western blots of both chicken gizzard membrane extracts and partially purified anion exchange fractions, but the antibody did not detect ecto-Apyrase in immunolabeled gizzard cryosections. In adult chicken stomach, the ecto-Apyrase was observed at the apical membrane of the glandular oxyntico-peptic cells as described in previous immunoperoxidase studies (Stout, J. G., R. S. Strobel, and T. L. Kirley (1995) Biochem. Mol. Biol. Int. 36, 529–535). However, ecto-ATPase was clustered in the sarcolemma of the organized layer of circular smooth muscle and in smooth muscle cells of the septa surrounding the glandular tissue, but not in the glandular cells containing the ecto-Apyrase. The findings indicate compartmentalization of the two related extracellular nucleotide hydrolyzing enzymes and suggest differential functions that are specialized for different regions of the chicken stomach. We also partially purified the ecto-Apyrase of chicken stomach, an 80-kDa membrane glycoprotein. Chicken stomach membranes were solubilized in digitonin, glycoproteins were separated from solubilized proteins by lectin chromatography, and nucleotide-binding glycoproteins were selected by immobilized Cibacron blue chromatography. Further purification by size exclusion and anion exchange chromatography yielded purification of 94-fold. The ATPase specific activity of the purified stomach ecto-Apyrase was 75,000 μmol of Pi/mg of protein/h, and the purified preparation consisted of a major band (55% of total protein) at 80 kDa. The purified enzyme could be deglycosylated with peptide N-glycosidase-F to a core molecular mass of 54 kDa. The N-terminal sequence of the 80-kDa stomach ecto-Apyrase band (which reacted with anti-ecto-ATPDase antibodies) was determined to be: MEYKGKVVAGLLTATWV. Immunological cross-reactivity data indicate that the stomach 80-kDa protein isolated is an ecto-Apyrase and is related to both the chicken liver and oviduct ecto-ATPDase enzymes characterized earlier, as well as to the human lymphoid cell activation antigen, CD39.
Gary Stacey - One of the best experts on this subject based on the ideXlab platform.
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enzymatic role for soybean ecto Apyrase in nodulation
Plant Signaling & Behavior, 2011Co-Authors: Kiwamu Tanaka, Tran Hong Nha Nguyen, Gary StaceyAbstract:Root nodulation is regulated by a variety of mechanisms. Ecto-Apyrase is an enzyme proposed to control the concentration of extracellular nucleotides. Transgenic expression of the soybean GS52 ecto-Apyrase was shown to stimulate nodulation. However, mutation of the enzyme to disrupt enzymatic activity prevented this effect. Therefore, the data suggest that the enzymatic activity of the ecto-Apyrase is critical for nodulation enhancement, suggesting a direct effect on extracellular nucleotide hydrolysis. In this article, we propose a hypothetical mechanism for plant ecto-Apyrase function during nodulation.
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enzymatic activity of the soybean ecto Apyrase gs52 is essential for stimulation of nodulation
Plant Physiology, 2011Co-Authors: Kiwamu Tanaka, Marc Libault, Cuong T Nguyen, Jianlin Cheng, Gary StaceyAbstract:Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-Apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 Apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 Apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation.
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gs52 ecto Apyrase plays a critical role during soybean nodulation
Plant Physiology, 2009Co-Authors: Manjula Govindarajulu, Gary Stacey, Sungyong Kim, Marc Libault, Howard R Berg, Kiwamu Tanaka, Christopher G TaylorAbstract:Apyrases are non-energy-coupled nucleotide phosphohydrolases that hydrolyze nucleoside triphosphates and nucleoside diphosphates to nucleoside monophosphates and orthophosphates. GS52, a soybean (Glycine soja) ecto-Apyrase, was previously shown to be induced very early in response to inoculation with the symbiotic bacterium Bradyrhizobium japonicum. Overexpression of the GS52 ecto-Apyrase in Lotus japonicus increased the level of rhizobial infection and enhanced nodulation. These data suggest a critical role for the GS52 ecto-Apyrase during nodulation. To further investigate the role of GS52 during nodulation, we used RNA interference to silence GS52 expression in soybean (Glycine max) roots using Agrobacterium rhizogenes-mediated root transformation. Transcript levels of GS52 were significantly reduced in GS52 silenced roots and these roots exhibited reduced numbers of mature nodules. Development of the nodule primordium and subsequent nodule maturation was significantly suppressed in GS52 silenced roots. Transmission electron micrographs of GS52 silenced root nodules showed that early senescence and infected cortical cells were devoid of symbiosome-containing bacteroids. Application of exogenous adenosine diphosphate to silenced GS52 roots restored nodule development. Restored nodules contained bacteroids, thus indicating that extracellular adenosine diphosphate is important during nodulation. These results clearly suggest that GS52 ecto-Apyrase catalytic activity is critical for the early B. japonicum infection process, initiation of nodule primordium development, and subsequent nodule organogenesis in soybean.
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Transgenic expression of the soybean Apyrase in Lotus japonicus enhances nodulation.
Plant physiology, 2005Co-Authors: Crystal Bickley Mcalvin, Gary StaceyAbstract:The soybean Apyrase, GS52, was previously characterized as an early nodulin that is expressed in roots and localized to the plasma membrane. Transgenic Lotus japonicus plants were constructed constitutively expressing the GS52 Apyrase. Segregation and Southern-blot analysis identified four single-copy sense lines, several double-copy sense lines, and one double-copy antisense line for further analysis. The single- and double-copy sense gs52 L. japonicus lines had enhanced nodulation that correlated with expression of the transgene. The sense transgenic lines were also found to have increased infection thread formation and enhanced infection zone length when infected by Mesorhizobium loti, the natural symbiont of L. japonicus. The data presented show that expression of the GS52 Apyrase can enhance nodulation in L. japonicus and points to an important role for this group of enzymes in nodulation.
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differential expression of two soybean Apyrases one of which is an early nodulin
Molecular Plant-microbe Interactions, 2000Co-Authors: Robert B Day, Roxanne Denny, Nevin D Young, C Mcalvin, John T Loh, T C Wood, Gary StaceyAbstract:Two cDNA clones were isolated from soybean (Glycine soja) by polymerase chain reaction with primers designed to conserved motifs found in Apyrases (nucleotide phosphohydrolase). The two cDNAs are predicted to encode for two, distinct, Apyrase proteins of approximately 50 kDa (i.e., GS50) and 52 kDa (i.e., GS52). Phylogenetic analysis indicated that GS52 is orthologous to a family of Apyrases recently suggested to play a role in legume nodulation. GS50 is paralogous to this family and, therefore, likely plays a different physiological role. Consistent with this analysis, GS50 mRNA was detected in root, hypocotyls, flowers, and stems, while GS52 mRNA was found in root and flowers. Neither gene was expressed in leaves or cotyledons. Inoculation of roots with Bradyrhizobium japonicum, nitrogen-fixing symbiont of soybean, resulted in the rapid (<6 h) induction of GS52 mRNA expression. The level of GS50 mRNA expression was not affected by bacterial inoculation. Western blot (immunoblot) analysis of GS50 expression mirrored the results obtained by mRNA analysis. However, in contrast to the mRNA results, GS52 protein was found in stems. Interestingly, anti-GS52 antibody recognized a 50-kDa protein found only in nodule extracts. Treatment of roots with anti-GS52 antibody, but not anti-GS50 antibody or preimmune serum, blocked nodulation by B. japonicum. Fractionation of cellular membranes in sucrose density gradients and subsequent Western analysis of the fractions revealed that GS50 colocalized with marker enzymes for the Golgi, while GS52 colocalized with marker enzymes for the plasma membrane. Restriction fragment length polymorphism (RFLP)-based mapping placed the gs52 gene on major linkage group J of the integrated genetic map of soybean. These data suggest that GS50 is likely an endo-Apyrase involved in Golgi function, while GS52 is localized on the root surface and appears to play an important role in nodulation.
Guido Guidotti - One of the best experts on this subject based on the ideXlab platform.
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widespread expression of ecto Apyrase cd39 in the central nervous system
Brain Research, 1998Co-Authors: Tingfang Wang, Guido GuidottiAbstract:We have shown that ecto-Apyrase protein is expressed in primary neurons and astrocytes in cell culture (T.-F. Wang, P.A. Rosenberg, G. Guidotti, 1997. Mol. Brain Res. 1997, 47: 295-302). Here we present immunohistochemical studies showing that ecto-Apyrase protein is widely distributed in rat brain, as it is present in neurons of the cerebral cortex, hippocampus and cerebellum as well as in glial cells and endothelial cells. Ecto-Apyrase is enriched in brain postsynaptic density membrane fractions and is localized in proximity to synaptophysin, the marker of synaptic vesicles. These results together with the observation that P2 purinergic receptors are present throughout the brain suggest that ecto-Apyrase is involved in regulating synaptic transmission mediated by extracellular ATP.
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characterization of brain ecto Apyrase evidence for only one ecto Apyrase cd39 gene
Molecular Brain Research, 1997Co-Authors: Tingfang Wang, Paul A Rosenberg, Guido GuidottiAbstract:A rat brain cDNA coding for ecto-(Ca,Mg)-Apyrase activity was isolated using human CD39 cDNA and functionally expressed in COS-7 cells. The gene codes for a protein with high similarity to human (75% identity) and murine (90% identity) CD39. It is expressed in primary neurons and astrocytes in cell culture as well as in kidney, liver, muscle and spleen. Southern analysis of the mouse genome suggests that there may be a single copy of the ecto-Apyrase gene. Interestingly, the human CD39 gene cytologically co-localizes with the susceptibility gene involved in human partial epilepsy with audiogenic symptoms; such a coincidence is consistent with reports on the deficiency of ecto-Apyrase activity in the brains of humans with temporal lobe epilepsy and in those of mice with audiogenic seizures.
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cd39 is an ecto ca2 mg2 Apyrase
Journal of Biological Chemistry, 1996Co-Authors: Tingfang Wang, Guido GuidottiAbstract:CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca2+,Mg2+)-Apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression and ecto-Apyrase activity on immune cells suggests that CD39 may be an ecto-Apyrase. This supposition is supported by the observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide triphosphatases. Finally, we show that CD39 indeed has ecto-Apyrase activity by expression in COS-7 cells.
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purification and cloning of a soluble atp diphosphohydrolase Apyrase from potato tubers solanum tuberosum
Biochemical and Biophysical Research Communications, 1996Co-Authors: Masahisa Handa, Guido GuidottiAbstract:A soluble ATP-diphosphohydrolase (Apyrase, EC 3.6.1.5) has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato Apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (Apyrase conserved regions) 1-4 that are highly conserved in known Apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed.
Jose M C Ribeiro - One of the best experts on this subject based on the ideXlab platform.
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Saliva of the soft tick, Ornithodoros moubata, contains anti-platelet and Apyrase activities.
Comparative Biochemistry and Physiology Part A: Physiology, 2003Co-Authors: Jose M C Ribeiro, Theresa M Endris, Richard G. EndrisAbstract:Abstract 1. 1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. 2. Saliva contains Apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca2+ and Mg2+ activate the reactions. 3. 3. The mean specific activities for ATP and ADP hydrolysis at pH7.5 were 0.97 and 0.74/moles orthophosphate/min/mg protein. 4. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary Apyrase activity have evolved independently in hematophagous arthropods.
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the salivary Apyrase of the blood sucking sand fly phlebotomus papatasi belongs to the novel cimex family of Apyrases
The Journal of Experimental Biology, 2001Co-Authors: Jesus G Valenzuela, Yasmine Belkaid, Edgar Rowton, Jose M C RibeiroAbstract:Apyrases are enzymes that hydrolyze nucleotide di- and triphosphates to orthophosphate and mononucleotides. At least two families of enzymes, belonging to the 5′-nucleotidase and to the actin/heat shock 70/sugar kinase superfamily, have evolved independently to serve the Apyrase reaction. Both families require either Ca(2+) or Mg(2+) for their action. A novel Apyrase enzyme sequence, with no homology to any other known protein sequence, was found recently in the salivary glands of the hematophagous bed bug Cimex lectularius. This enzyme functions exclusively with Ca(2+). Here, we report the finding of a cDNA similar to that of the C. lectularius salivary Apyrase isolated from a salivary gland cDNA library of Phlebotomus papatasi. Transfection of insect cells with the P. papatasi salivary gland Apyrase cDNA resulted in the secretion of a Ca(2+)-dependent Apyrase whose activity was indistinguishable from that in salivary homogenates of P. papatasi. Homologous sequences were found in humans, in another sand fly (Lutzomyia longipalpis), in the fruit fly Drosophila melanogaster, in the nematode Caenorhabditis elegans and in the protozoan Cryptosporidium parvum, indicating that this family of enzymes is widespread among animal species.
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toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly lutzomyia longipalpis
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Rosane Charlab, Jesus G Valenzuela, Edgar D Rowton, Jose M C RibeiroAbstract:The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius Apyrase, (ii) a 5′-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the blast databases. Following these findings, we observed that the salivary Apyrase activity of L. longipalpis is indeed similar to that of Cimex Apyrase in its metal requirements. The predicted isoelectric point of the putative Apyrase matches the value found for Lutzomyia salivary Apyrase. A 5′-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to α-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.
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purification cloning and expression of an Apyrase from the bed bug cimex lectularius a new type of nucleotide binding enzyme
Journal of Biological Chemistry, 1998Co-Authors: Jesus G Valenzuela, Rosane Charlab, Michael Y Galperin, Jose M C RibeiroAbstract:An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying Apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purified C. lectularius Apyrase was an acidic protein with a pI of 5.1 and molecular mass of approximately 40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectularius cDNA corresponding to the N-terminal sequence of purified Apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with native C. lectularius Apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized both C. lectularius Apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the Apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent Apyrase with a pI of 5.1 and immunoreactive material detected by the anti-Apyrase serum. C. lectularius Apyrase has no significant sequence similarity to any other known Apyrases, but homologous sequences have been found in the genome of the nematode C. elegans and in mouse and human expressed sequence tags from fetal and tumor EST libraries.
Stanley J Roux - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of arabidopsis Apyrase family reveals their roles in regulating endomembrane ndp nmp homoeostasis
Biochemical Journal, 2015Co-Authors: Stanley J Roux, Tsanyu Chiu, Jeemeng Lao, Bianca Manalansan, Dominique Loque, Joshua L HeazlewoodAbstract:Plant Apyrases are nucleoside triphosphate (NTP) diphosphohydrolases (NTPDases) and have been implicated in an array of functions within the plant including the regulation of extracellular ATP. Arabidopsis encodes a family of seven membrane bound Apyrases (AtAPY1-7) that comprise three distinct clades, all of which contain the five conserved Apyrase domains. With the exception of AtAPY1 and AtAPY2, the biochemical and the sub-cellular characterization of the other members are currently unavailable. In this research, we have shown all seven Arabidopsis Apyrases localize to internal membranes comprising the cis-Golgi, endoplasmic reticulum (ER) and endosome, indicating an endo-Apyrase classification for the entire family. In addition, all members, with the exception of AtAPY7, can function as endo-Apyrases by complementing a yeast double mutant (Δynd1Δgda1) which lacks Apyrase activity. Interestingly, complementation of the mutant yeast using well characterized human Apyrases could only be accomplished by using a functional ER endo-Apyrase (NTPDase6), but not the ecto-Apyrase (NTPDase1). Furthermore, the substrate specificity analysis for the Arabidopsis Apyrases AtAPY1-6 indicated that each member has a distinct set of preferred substrates covering various NDPs (nucleoside diphosphates) and NTPs. Combining the biochemical analysis and sub-cellular localization of the Arabidopsis Apyrases family, the data suggest their possible roles in regulating endomembrane NDP/NMP (nucleoside monophosphate) homoeostasis.
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Apyrase suppression raises extracellular atp levels and induces gene expression and cell wall changes characteristic of stress responses
Plant Physiology, 2014Co-Authors: Min Hui Lim, Greg Clark, Jianchao Yao, Ignacio F Gallardo, Jason W Dugger, Lauren J Webb, James N Huang, Mari L Salmi, Jawon Song, Stanley J RouxAbstract:Plant cells release ATP into their extracellular matrix as they grow, and extracellular ATP (eATP) can modulate the rate of cell growth in diverse tissues. Two closely related Apyrases (APYs) in Arabidopsis (Arabidopsis thaliana), APY1 and APY2, function, in part, to control the concentration of eATP. The expression of APY1/APY2 can be inhibited by RNA interference, and this suppression leads to an increase in the concentration of eATP in the extracellular medium and severely reduces growth. To clarify how the suppression of APY1 and APY2 is linked to growth inhibition, the gene expression changes that occur in seedlings when Apyrase expression is suppressed were assayed by microarray and quantitative real-time-PCR analyses. The most significant gene expression changes induced by APY suppression were in genes involved in biotic stress responses, which include those genes regulating wall composition and extensibility. These expression changes predicted specific chemical changes in the walls of mutant seedlings, and two of these changes, wall lignification and decreased methyl ester bonds, were verified by direct analyses. Taken together, the results are consistent with the hypothesis that APY1, APY2, and eATP play important roles in the signaling steps that link biotic stresses to plant defense responses and growth changes.
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effects of chemical inhibitors and Apyrase enzyme further document a role for Apyrases and extracellular atp in the opening and closing of stomates in arabidopsis
Plant Signaling & Behavior, 2013Co-Authors: Greg Clark, Cameron Darwin, Viraj Mehta, Faith Jackobs, Tyler Perry, Katia Hougaard, Stanley J RouxAbstract:In Arabidopsis leaves there is a bi-phasic dose-response to applied nucleotides; i.e., lower concentrations induce stomatal opening, while higher concentrations induce closure. Two mammalian purinoceptor antagonists, PPADS and RB2, block both nucleotide-induced stomatal opening and closing. These antagonists also partially block ABA-induced stomatal closure and light-induced stomatal opening. There are two closely related Arabidopsis Apyrases, AtAPY1 and AtAPY2, which are both expressed in guard cells. Here we report that low levels of Apyrase chemical inhibitors can induce stomatal opening in the dark, while Apyrase enzyme blocks ABA-induced stomatal closure. We also demonstrate that high concentrations of ATP induce stomatal closure in the light. Application of ATPγS and chemical Apyrase inhibitors at concentrations that have no effect on stomatal closure can lower the threshold for ABA-induced closure. The closure induced by ATPγS was not observed in gpa1-3 loss-of-function mutants. These results furth...
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co regulation of exine wall patterning pollen fertility and anther dehiscence by arabidopsis Apyrases 6 and 7
Plant Physiology and Biochemistry, 2013Co-Authors: Jian Yang, Greg Clark, Jian Wu, Dwight K Romanovicz, Stanley J RouxAbstract:Abstract An NCBI nucleotide blast keyed to Apyrase (ATP-diphosphohydrolases, EC 3.6.1.5) conserved regions revealed five Apyrases, AtAPYs ( 3 - 7 ), in addition to the previously identified AtAPY1 and 2 . Here we report the functional analyses of two of the newly defined Apyrases, AtAPY6 and AtAPY7 . We analyzed tissue specificity of AtAPY6 and 7 expression by qRT-PCR and promoter:GUS fusion assays. We characterized the phenotypes of single and double knockout mutants for AtAPY6 and 7 in anther and pollen by light microscopy and electron microscopy. The transcripts of both AtAPY6 and 7 are expressed in mature pollen grains. Single knockout mutants of AtAPY6 and 7 displayed a minor change in pollen exine pattern under scanning electron microscopy without obvious change in fertility. Double knockout mutants of AtAPY6 and 7 ( apy6apy7 ) displayed severe defects in pollen exine pattern, deformed pollen shape and reduced male fertility. An analysis of pollen from heterozygous apy6apy7 plants suggests that the defects in pollen exine wall are determined by the diploid genome. Our findings demonstrate that AtAPY6 and AtAPY7 are enzymes that play an important role in exine development of pollen grains, possibly through regulating the production of key polysaccharides needed for proper assembly of the exine layer.
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multiherbicide tolerance conferred by atpgp1 and Apyrase overexpression in arabidopsis thaliana
Nature Biotechnology, 2003Co-Authors: Brian Windsor, Stanley J Roux, Alan C LloydAbstract:Herbicide resistance is an important trait often introduced into crop plants. Mechanisms of resistance can involve a mutant target protein that is unaffected by the herbicide, or metabolic detoxification or degradation of the herbicide. Recently, we showed that overexpression in Arabidopsis thaliana of either psNTP9, the garden pea Apyrase gene, or AtPgp1, the A. thaliana homolog of the plant multidrug resistance (MDR) gene, enabled A. thaliana to germinate on the toxin cycloheximide and to grow better on toxic levels of the plant hormone N6-[2-isopentyl]adenine (2iP). Here we report that overexpression of either MDR or Apyrase proteins resulted in increased resistance to herbicides from different chemical classes. Apyrase inhibition by small molecule inhibitors reversed this resistance. Treatment of untransformed plants with an Apyrase inhibitor increased their sensitivity to the same herbicides. These results indicate that the genes may be involved in a resistance mechanism relating to decreased retention or increased active efflux of herbicide from the plant cell.