Human placental atp-diphosphohydrolase: Biochemical characterization, regulation and function

Kettlun, Ana M.; Álvarez, Alejandra; Quintar, R.; Valenzuela, Manuel; Collados, L.; Aranda, Evelyn; Banda, Adam; Chayet, L.; Chiong, Mario; Mancilla, Marta; Traverso-Cori, A.

doi:10.1016/0020-711x(94)90065-5

Cited by 34 publications

(17 citation statements)

References 37 publications

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“…A recent study by Kettlun et al [40] deals with the properties of human placental ATP-DPH. This preparation was obtained after isoelectric focusing of solubilized microsomes and elution of the enzymic activity.…”

Section: Discussionmentioning

confidence: 99%

Purification and Properties of Human Placental ATP Diphosphohydrolase

Christoforidis¹,

Papamarcaki²,

Galaris³

et al. 1995

European Journal of Biochemistry

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ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, 0. (1990) Mol. Cell. Biochern. 97, 1-81, 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-[a-"PP]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not nionophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca" or Mg". The K,,, values for ATP and ADP were determined to be 10 pM and 20 pM, 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 NaN,, NaF, adenosine S'-[/)',;I-imido]triphosphate and adenosine 5'-[ n,/Amethylene 1 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.

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Section: Discussionmentioning

confidence: 99%

Purification and Properties of Human Placental ATP Diphosphohydrolase

Christoforidis¹,

Papamarcaki²,

Galaris³

et al. 1995

European Journal of Biochemistry

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“…Later, ATP diphosphohydrolase (or apyrase) was identified in human term placenta [81,198,316]. Two isoforms (enzymes I and II) of placental ecto-ATP diphosphohydrolases were later identified by cDNA cloning [263].…”

Section: Placentamentioning

confidence: 99%

Purinergic signalling in the reproductive system in health and disease

Burnstock

2013

Purinergic Signalling

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There are multiple roles for purinergic signalling in both male and female reproductive organs. ATP, released as a cotransmitter with noradrenaline from sympathetic nerves, contracts smooth muscle via P2X1 receptors in vas deferens, seminal vesicles, prostate and uterus, as well as in blood vessels. Male infertility occurs in P2X1 receptor knockout mice. Both short-and long-term trophic purinergic signalling occurs in reproductive organs. Purinergic signalling is involved in hormone secretion, penile erection, sperm motility and capacitation, and mucous production. Changes in purinoceptor expression occur in pathophysiological conditions, including pre-eclampsia, cancer and pain.

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“…ATP diphosphohydrolase (apyrase) is an enzyme able to promote the removal of two phosphate groups of ATP but of only one phosphate group of ADP. This enzyme presents divalent cation dependence and can be stimulated by Ca 2+ and Mg 2+ (12,15,30,32). The ecto-5-nucleotidase can also be stimulated by Mg 2+ (14,31) but this activation was lower than the apyrase activation (26.3 ± 5% for the ecto-5-nucleotidase activation and 1490 ± 84% for the apyrase activation in relation to the control).…”

Section: Atp Adp and Amp Hydrolysismentioning

confidence: 99%

“…To show that ATP and ADP hydrolysis occurs due to an apyrase and that one active site is able to hydrolyze the two substrates, we used the Chevillard competition plot used by Kettlun et al (32) to characterize a human placental ATP diphosphohydrolase. To assay the combination of substrate concentrations in a Chevillard competition plot (26) we chose concentrations at which the rate of hydrolysis was the same when either ATP or ADP was used as substrate.…”

Section: Atp Adp and Amp Hydrolysismentioning

confidence: 99%

Ectonucleotidase activities in Sertoli cells from immature rats

Casali

Silva

Gelain

et al. 2001

Braz J Med Biol Res

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Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P 2Y2 and P A1 . The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri-and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 ± 6 and 21 ± 2 nmol Pi mg -1 min -1 for ATP and ADP, respectively). The ecto-5-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 ± 2 nmol Pi mg -1 min -1 for AMP and 1.52 ± 0.13 nmol adenosine mg -1 min -1 , respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.

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Human placental atp-diphosphohydrolase: Biochemical characterization, regulation and function

Cited by 34 publications

References 37 publications

Purification and Properties of Human Placental ATP Diphosphohydrolase

Purification and Properties of Human Placental ATP Diphosphohydrolase

Purinergic signalling in the reproductive system in health and disease

Ectonucleotidase activities in Sertoli cells from immature rats

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