Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mimosa pudica Linn leaves with pulvini contain unique isoforms (I and II) of Apyrase enzyme (EC 3.6.1.5). The activity of isoform I depends on divalent cation Mn2+. This isoform is associated noncovalently with the polysaccharide, containing mainly of galactose and arabinose sugars. The apparent molecular mass of these 2 isoforms are 36 and 34 Kd respectively. The association of the polysaccharide with the isoform I has been found to be Ca2+ dependent which is endogenously present in this isoform. Removal of Ca2+ and polysaccharide from the enzyme (isoform I) leads to an inactivation. The enzyme activity can be restored when both Ca2+ and endogenous polysaccharide fraction were added at an optimal molar ratio of Ca2+:protein of 7:1. The endogenous polysaccharide can be replaced by the standard arabinogalactan. No other sugar or polysaccharide except the arabinogalactan can restore the apyrase activity. Calcium mediates a conformational change in the protein which helps in association of polysaccharide as evidenced from fluorometric and far UV-CD studies to restore the enzymic activity. Neither any interaction of the polysaccharide with the protein is detected in absence of Ca2+ nor the enzyme activity could be recovered under such condition.
Mol Cell Biochem 1998 Oct
PMID:Mimosa pudica apyrase requires polysaccharide and Ca2+ for the activity. 978 42

The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252-29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes.
Mol Biochem Parasitol 1998 Nov 30
PMID:Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase. 987 99

Apyrase activity (ATP diphosphohydrolase, EC 3.6.1.5) was detected in salivary glands of the cat flea Ctenocephalides felis. Whole extracts of salivary glands contain approximately 21 ng of protein, 145 U/mg ADP'ase and 158 U/mg ATP'ase activity; AMP is not hydrolysed by salivary gland extracts. DEAE-Sepharose CL-6B anion exchange chromatography, and Cibacron Blue affinity chromatography each give a single coincident peak of ADP/ATP'ase activity. Biogel P-100 gel filtration of salivary gland homogenates made in buffer containing Triton and protease inhibitors, separated enzymatic activity into 57 kD and 44 kD peaks of ADP/ATP'ase activity. Partially purified ADP/ATP'ases are dependent on divalent cations and activation increases between 0.125 mM and 5.0 mM calcium. At 5 mM, magnesium is almost equally effective as calcium in activating ADP/ATP'ase but manganese and zinc are less so, and EDTA abolishes activity. ADP/ATP'ases have a pH optima of 7-9. The Km for ADP hydrolysis by whole extracts and partially purified enzyme is approximately 66 microM ADP. The co-purification of ADP'ase and ATP'ase activity by three physiochemical techniques and parallelism between ADP and ATP hydrolysis under varying conditions of pH and activating cation indicates enzymatic activity is attributable to true apyrase(s).
Insect Biochem Mol Biol 1998 Dec
PMID:Characterization of apyrase activity from the salivary glands of the cat flea Ctenocephalides felis. 988 18

Recent studies from our laboratory have found that a root lectin from the legume Dolichos hifloris is present on the root surface, binds rhizobial Nod factor and has apyrase activity. To assess the broader significance of this lectin/nucleotide phosphohydrolase (Db-LNP), we have cloned a second related cDNA (Db-apyrase-2) from D. hiflorus, as well as related cDNAs from the legumes Lotus japonicus and Medicago sativa, and from Arabidopsis thaliana, a non-legume. The deduced amino acid sequences of these apyrases were aligned with one another and with the sequences of other apyrases from plants, animals, yeast and protozoa. Phylogenetic analysis shows that Db-LNP has closely related orthologs only in other legumes, while Db-apyrase-2 is more closely related to apyrase sequences from non-leguminous plants. We also show that the orthologs of Db-LNP from M. sativa and Pisum sativum have carbohydrate binding activity. The results suggest that legume LNPs may represent a special class of apyrases that arose by gene duplication and subsequent specialization.
Mol Gen Genet 1999 Sep
PMID:A Nod factor-binding lectin is a member of a distinct class of apyrases that may be unique to the legumes. 1051 21

Mosquito salivary proteins, which are fundamental to the process of blood feeding, also facilitate disease transmission and cause allergic reactions. The identification and characterisation of these proteins have been hampered by the difficulty of obtaining them in purified form. In this report, we describe the production of mouse monoclonal antibodies (mAbs) against mosquito salivary proteins. BALB/c mice were immunised with Aedes aegypti saliva proteins. Hybridomas were produced by fusion of spleen cells with a mouse myeloma cell line. Positive clones were selected using a saliva-capture ELISA and further identified using immunoblotting. Three mAbs reacted with a 44 kDa protein (Aed a X1) in the saliva-immunoblotting, and did not react with 2 recombinant salivary proteins, rAed a 1 (apyrase) and rAed a 2 (D7), in both immunoblotting and ELISA. Two other mAbs reacted with a 37 kDa protein in saliva-immunoblotting, but failed to react with the 37 kDa rAed a 2 in either immunoblotting or ELISA, suggesting that there is a second 37 kDa protein (Aed a X2) which is recognised by the two mAbs. The 44 kDa and 37 kDa proteins have not been previously identified. These mAbs provide a means to purify proteins, to isolate new genes from the salivary gland cDNA library, and to standardise mosquito extracts, facilitating studies of disease transmission by mosquitoes and of mosquito allergy.
Insect Biochem Mol Biol 1999 Oct
PMID:Production and characterization of monoclonal antibodies to two new mosquito Aedes aegypti salivary proteins. 1052 10

Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and approximately 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the filament.
J Mol Biol 2000 Jan 21
PMID:Polymerization and structure of nucleotide-free actin filaments. 1062 43

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.
Mol Biol Cell 2000 May
PMID:Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26. 1079 54

Cell fusion is a central phenomenon during the immune response that leads to formation of large elements called multinucleated giant cells (MGCs) of common occurrence at sites of granulomatous inflammation. We have previously reported on the involvement in this event of a novel receptor expressed to high level by mononuclear phagocytes, the purinergic P2X(7) receptor. Herein, we show that blockade of this receptor by a specific monoclonal antibody prevents fusion in vitro. In contrast, cell fusion is stimulated by addition of enzymes that destroy extracellular ATP (i.e., apyrase or hexokinase). Experiments performed with phagocytes selected for high (P2X(7) hyper) or low (P2X(7) hypo) P2X(7) expression show that fusion only occurs between P2X(7) hyper/P2X(7) hyper and not between P2X(7) hyper/P2X(7) hypo or P2X(7) hypo/P2X(7) hypo. During MGCs formation we detected activation of caspase 3, an enzyme that is powerfully stimulated by P2X(7). Finally, we observed that during MGCs formation, the P2X(7) receptor is preferentially localized at sites of cell-to-cell contact. These findings support the hypothesis originally put forward by our group that the P2X(7) receptor participates in multinucleated giant cell formation.
Mol Biol Cell 2000 Sep
PMID:P2X(7) receptor and polykarion formation. 1098 8

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca(2+) signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca(2+) waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20-40 min. The peptides were shown to be more effective in inhibiting Ca(2+) waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca(2+) waves by connexin mimetic peptides did not affect the Ca(2+) response to extracellular ATP. Although the intracellular Ca(2+) response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca(2+) signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca(2+) wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.
Am J Physiol Lung Cell Mol Physiol 2000 Oct
PMID:Connexin mimetic peptides reversibly inhibit Ca(2+) signaling through gap junctions in airway cells. 1100 Jan 20

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.
Mol Plant Microbe Interact 2000 Oct
PMID:Differential expression of two soybean apyrases, one of which is an early nodulin. 1104 67


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>