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Kinetic properties of E-NTPDase activity in lymphocytes isolated from bone marrow, thymus and mesenteric lymph nodes of Wistar rats

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Abstract

Plasma membrane anchored nucleotidases (E-ATPDases), as the E-NTPDase family, could hydrolyze and regulate the pericellular levels of nucleotides in lymphocytes. Each immune organ has a different microenvironment and display lymphocytes with different functions and phenotypes. Considering the different functions of each resident subpopulations of lymphocytes, the E-ATPDases activities in bone marrow (BML), thymus (TL) and mesenteric lymph node (MLL) lymphocytes of Wistar rats were characterized. The hydrolysis of extracellular nucleotides (eATP and eADP) showed linearity in time of reaction between 0 and 120 min, and concentration of lymphocytes expressed in proteins between 1 and 6 μg protein in the reaction medium. The optimal activity was attained at 37 °C in a pH value of 8.0. The necessity of the cofactors Ca2+ and Mg2+ for the enzymatic activity was confirmed through a curve of concentration of 0–1000 µM in the reaction medium, with both cofactors showing similar effects in the enzymatic activity. The Chevillard plot revealed that the hydrolysis of eATP and eADP occurred at the same active site of the enzyme. The analyses of E-ATPDases inhibitor and enzyme specificity showed possible involvement of E-NTPDase isoforms − 1 and − 2 in the isolated cells. Furthermore, different kinetic behavior of the nucleotide hydrolysis in each resident subpopulation lymphocyte was observed in this study, as MLL showed the higher Vmax with the lowest km values, while TL had the lowest Vmax and high km values. The kinetic values for E-NTPDase activity of each immune tissue lymphocytes can be an important therapeutic target for numeral immune-related diseases.

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Data availability

The authors declare that all data were generated in-house and that no paper mill was used. The authors declare that the datasets generated and analyzed during the current study support the findings of the study and are available within the article. All authors have agreed to submit the manuscript solely to your journal. Data available on request from the authors. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

eATP:

Extracellular adenosine triphosphate

eADP:

Extracellular adenosine diphosphate

eADO:

Extracellular adenosine

E-NTPDase:

Ectonucleoside triphosphate diphosphohydrolase

BM:

Bone marrow

BML:

Bone marrow lymphocytes

TL:

Thymus lymphocytes

ML’s:

Mesenteric lymph nodes

MLL:

Mesenteric lymph node lymphocytes

Vmax :

Maximum enzyme activity

km :

Michaelis–Menten constant

P2X/P2Y:

Purinergic receptors

LGL:

Large granular lymphocytes

SML:

Small lymphocytes

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Funding

We gratefully acknowledge the financial support provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROEX/CAPES, Grant No. 23038004173/2019-93) for the work and scholarship provided for Pedro Henrique Doleski by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) and Capes PrInt (Process No. 88887.357232/2019-00).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Universidade Federal de Santa Maria, Santa Maria, Brazil

    Pedro Henrique Doleski, Matheus Henrique Jantsch, Renan Silva Ebone, Stephen Adeniyi Adefegha, Daniela Bitencourt Rosa Leal & Maria Rosa Chitolina Schetinger

  2. Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, Brazil

    Pedro Henrique Doleski, Fernanda Licker Cabral & Daniela Bitencourt Rosa Leal

  3. Department of Biochemistry, Federal University of Technology, Akure, Nigeria

    Stephen Adeniyi Adefegha

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  1. Pedro Henrique Doleski
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  2. Fernanda Licker Cabral
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Contributions

Conceptualization: PHD, MRCS, DBRL; Methodology: PHD, FLC, MHJ, RSE; Formal analysis and investigation: PHD; Writing—original draft preparation: PHD, SAA; Writing—review and editing: MRCS, DBRL, SAA; Supervision: MRCS.

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Correspondence to Pedro Henrique Doleski.

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The authors have declared no conflict of interest.

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The Animal Ethics Committee from Universidade Federal de Santa Maria, Brazil (protocol under No. 6788030518) approved the study.

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Doleski, P.H., Cabral, F.L., Jantsch, M.H. et al. Kinetic properties of E-NTPDase activity in lymphocytes isolated from bone marrow, thymus and mesenteric lymph nodes of Wistar rats. Mol Cell Biochem (2023). https://doi.org/10.1007/s11010-023-04860-7

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