Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported. A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor. Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.
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PMID:New insights on P2X purinoceptors. 905 29

ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
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PMID:Mg-dependent ecto-ATPase activity in Leishmania tropica. 914 51

Kinetic analysis of enzymatic hydrolysis of 14C- or 3H-labelled ATP was performed in the blood of healthy men donors and male Wistar rats. Nonspecific phosphatases were paralleled in blood serum by specific enzyme ATPase which is capable of dephosphorylating exogenous ATP in cooperation with other serum nucleotidases ultimately to adenosine. APparent Michaelis-Menten constant (Km) and maximal velocity (Vmax) values for rat serum ATPases are 68 +/- 7 microM and 7.0 +/- 0.3 nmoles ATP/mg protein per h, respectively. ATPase from human blood serum was characterized by lower respective Km and Vmax values: 39 +/- 5 microM and 2.5 +/- 0.2 nmoles ATP/mg protein per h. Activity of serum ATPase was decreased in the presence of membrane ecto-ATPase inhibitors PPADS and RB2, whereas the Na,K-ATPase inhibitor ouabain did not exert any inhibitory action on the measured enzyme activity.
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PMID:Kinetic analysis of enzymatic hydrolysis of ATP in human and rat blood serum. 928 43

We have examined the in vivo localization 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 micromol 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.
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PMID:Immunolocalization of the ecto-ATPase and ecto-apyrase in chicken gizzard and stomach. Purification and N-terminal sequence of the stomach ecto-apyrase. 929 5

Ecto-ATPase activity was measured for red blood cells, white blood cells, and whole blood from a variety of vertebrates. A large range of red blood cell ecto-ATPase activity was observed; for example, at 10 degrees C, red blood cells from a catastomid fish (Catostomus macrocheilus) and a newt (Taricha rivularis) had activities of 56 +/- 9 and 25,000,000 +/- 14,000,000 pmol ATP per 10(6) red blood cells per hour, respectively (mean +/- SD). Several control experiments verified that the measured ATPase activity was not the result of intracellular ATPases released due to cell damage or lysis nor due to the release of intracellular nucleoside triphosphate or uptake of extracellular ATP. Red blood cell ecto-ATPase activity was relatively low within the teleosts, was high within the reptiles, and had the greatest range and single highest value within the amphibians. Within the endotherms, avian red blood cell ecto-ATPase activities were greater than mammalian red blood cell ecto-ATPase activities, which were the lowest for all vertebrates examined. The lowest ecto-ATPase activities measured were for human and skunk red blood cells, which had activities of 13 +/- 1 and 11 +/- 2 pmol ATP per 10(6) red blood cells per hour, respectively, at 35 degrees C. Ecto-ATPase activity was measured in white blood cells of several vertebrate species and appeared generally high and less variable than red blood cell ecto-ATPase activity. Measured whole blood ecto-ATPase activity showed a range of three orders of magnitude and correlated positively with red blood cell ecto-ATPase activities. Ecto-ATPase activity was also determined for red blood cells from fetal, 1-3 d old neonatal, and pregnant garter snakes (Thamnophis elegans); these activities were not significantly different from the activity of red blood cells from nonpregnant adult females. Overall, the data from the present study demonstrate a wide range of red blood cell and whole blood ecto-ATPase activities among vertebrates and include some of the highest ecto-ATPase activities reported to date.
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PMID:Ecto-ATPase activity of vertebrate blood cells. 936 Nov 36

Considerable evidence indicates that ATP, acting intracellularly of as a neurotransmitter, can influence nerve cell physiology in a variety of ways. Defects in the functioning of ATP-metabolizing enzymes could therefore lead to disturbances in neurotransmission and creation of sustained neuronal discharges characteristic of status epilepticus. In this study we investigated synaptosomal ATPase changes in rat brains during lithium/pilocarpine-induced status epilepticus. After 2 h of continuous electroencephalographic spiking, both Mg(2+)- and Ca(2+)-dependent ecto-ATPases were significantly decreased in freshly prepared synaptosomal preparations from the status rats. The intracellularly acting Ca2+Mg(2+)-ATPase (Ca-pump) was also decreased, but no changes occurred in synaptosomal Na+K(+)-ATPase activity. The difference between ecto-ATPase activities of the control and status rat brains was not affected by repeated freezing-thawing and lengthy storage. Possible involvement of reduced synaptosomal divalent cation-dependent ATPases in the pathophysiology of status epilepticus is discussed.
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PMID:Reduced cortical ecto-ATPase activity in rat brains during prolonged status epilepticus induced by sequential administration of lithium and pilocarpine. 937 20

The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolyzed added ATP and this reaction was inhibited by suramin and RB2. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM. We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.
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PMID:Involvement of ecto-ATPase and extracellular ATP in polymorphonuclear granulocyte-endothelial interactions. 951 66

We have investigated the effect of cross-linking on the enzymatic activity and oligomer formation of the chicken stomach ecto-apyrase. Cross-linking with the hydrophobic, lysine-specific dithiobis(succinimidylpropionate) (DSP) caused equal inhibition of ATPase and ADPase activity in both the membrane-bound and detergent-solubilized ecto-apyrase. The inhibitory effect of cross-linking was reversed upon the addition of the reductant dithiothreitol. Western blots of aliquots of the cross-linked samples show decreased amounts of the monomeric 80 kDa ecto-apyrase and the appearance of a 160 kDa dimer under conditions inducing enzyme inhibition. Therefore, the chicken stomach ecto-apyrase, like the chicken gizzard ecto-ATPase, is likely a homodimer in vivo. Unlike the related gizzard ecto-ATPase, however, the native stomach ecto-apyrase is not stimulated, but rather inhibited by cross-linking, presumably due to different quaternary structural stability of the two enzymes.
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PMID:Cross-linking induces homodimer formation and inhibits enzymatic activity of chicken stomach ecto-apyrase. 955 6

This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.
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PMID:The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea. 958 Apr 35

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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PMID:Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases1. 967 46


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