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Biochemical characterization of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat heart left ventricle

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Abstract

In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence and the K M value corresponded to 91.42 ± 13.97 μM and the maximal velocity (V max ) value calculated was 63.79 ± 3.59 nmol p-nitrophenol released/min/mg of protein (mean ± SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5′-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5′-TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had no effects on p-Nph-5′-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated the expression of NPP2 and NPP3 enzymes, but excluded the presence of NPP1 member. By quantitative real-time PCR we identified the NPP3 as the enzyme with the highest expression in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that are involved in several cardiac pathologies.

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Aknowledgments

We thank Dr. Maria Luiza Morais de Barreto-Chaves for providing Collagenase type II. We thank Dr. Arthur Germano Fett-Neto and his students for helping us with the gel pictures. This work was supported by grants and fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Authors and Affiliations

  1. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul – UFRGS, Rua Ramiro Barcelos, 2600 – Prédio ANEXO, Porto Alegre, RS, CEP 90035-003, Brazil

    Bárbara Rücker, Manoela E. Almeida & João José Freitas Sarkis

  2. BIDMC Genomics Center and Harvard Medical School, Boston, MA, USA

    Towia A. Libermann & Luiz F. Zerbini

  3. Departamento de Ciências Fisiológicas, Fundação Faculdade Federal de Ciências Medicas de Porto Alegre – FFFCMPA, Porto Alegre, RS, Brazil

    Márcia R. Wink

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  1. Bárbara Rücker
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  2. Manoela E. Almeida
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  3. Towia A. Libermann
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  4. Luiz F. Zerbini
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  5. Márcia R. Wink
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  6. João José Freitas Sarkis
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Correspondence to João José Freitas Sarkis.

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Rücker, B., Almeida, M.E., Libermann, T.A. et al. Biochemical characterization of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat heart left ventricle. Mol Cell Biochem 306, 247–254 (2007). https://doi.org/10.1007/s11010-007-9576-5

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