Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

Kukulski, Filip; Komoszyński, Michał

doi:10.1046/j.1432-1033.2003.03734.x

Cited by 57 publications

(50 citation statements)

References 43 publications

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“…An NTPDase purified from porcine brain has K m for ATP and ADP of 97 and 95 mM, respectively (Kukulski and Komoszynski, 2003). Biochemical properties of recombinant GS52 were also reported (Tanaka et al, 2011).…”

Section: Discussionmentioning

confidence: 97%

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

et al. 2011

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We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.

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Section: Discussionmentioning

confidence: 97%

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

et al. 2011

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“…ATP is metabolized to ADP, AMP and adenosine by ecto-ATPases and ectonucleotidases and the latter reaches levels of 50-80 nmole/liter at rest Kukulski and Komoszynski, 2003). Adenosine that comes from the breakdown of nucleotides or other sources is an inhibitor of baseline or resting Cl − secretion and can mask some of the secretory effects of excitatory mediators that are released spontaneously .…”

Section: Sensors: Enterochromaffin Cellsmentioning

confidence: 99%

Autonomic nervous system and secretion across the intestinal mucosal surface

Xue

Askwith

Javed

et al. 2007

Autonomic Neuroscience

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Chloride secretion is important because it is the driving force for fluid movement into the intestianl lumen. Flow of accummulated fluid flushes out invading micro-orgnaisms in defense of the host. Chloride secretion is regulated by neurons in the submucosal plexus of the enteric nervous system. Mechanosensitive enterochromaffin cells that release 5-hydroxytryptamine (5-HT)activate intrinsic afferent neurons in the submucosal plexus and initiate chloride secretion. Mechanical stimulation by distention may also triggers reflexes by a direct action on intrinsic afferent neurons. Dysregulation of 5-HT release or altered activity of intrinsic afferents is likely to occur in states of inflammation and other disorders.

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“…E-NTPDase1 rapidly converts both ATP and ADP to AMP, thereby depleting the extracellular space of ligands to P2X and P2Y receptors. E-NTPDase1 has an at least three times lower Michaelis constant than the other members of this enzyme family [9], making it an ideal candidate to terminate P2-receptor-mediated signaling or prevent the inactivation of purinergic receptors (for review, see [10]). ENTPDase2, expressed by astrocytes, converts preferentially ATP into ADP but has a much lower hydrolysis rate for ADP [11], which can lead to ADP accumulation.…”

Section: Introductionmentioning

confidence: 99%

NTPDase1 activity attenuates microglial phagocytosis

Bulavina

Szulzewsky

Rocha

et al. 2012

Purinergic Signalling

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Purinergic signaling plays a major role in the regulation of phagocytosis in microglia. Interplay between P2 and P1 receptor activation is controlled by a cascade of extracellular enzymes which dephosphorylate purines resulting in the formation of adenosine. The ATP-and ADP-degrading capacity of cultured microglia depends on the expression of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) and is several times higher when compared to astrocytes which lack this enzyme. In brain slices, deletion of CD39 resulted in a 50 % decrease of ADPdegrading ability, while the degradation of ATP was decreased to about 75 % of the values measured in wild-type brain tissue. Microglia in acute slices from cd39 −/− animals had increased constitutive phagocytic activity which could not be further enhanced by ATP in contrast to control animals. Pharmacological blockage of P2 receptors decreased the constitutive phagocytic activity to a similar base level in wild-type and cd39 −/− microglia. Activation of P1receptors by non-hydrolysable adenosine analog significantly decreased phagocytic activity. Deletion of CD73, an enzyme expressed by microglia which converts AMP to adenosine did not affect phagocytic activity. Taken together, these data show that CD39 plays a prominent role in controlling ATP levels and thereby microglial phagocytosis.

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Purification and characterization of NTPDase1 (ecto‐apyrase) and NTPDase2 (ecto‐ATPase) from porcine brain cortex synaptosomes

Cited by 57 publications

References 43 publications

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

Purification and Biochemical Characterization of a Novel Ecto-Apyrase, MP67, from Mimosa pudica

Autonomic nervous system and secretion across the intestinal mucosal surface

NTPDase1 activity attenuates microglial phagocytosis

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