Caracterização enzimática da e-ntpdase em linfócitos residentes de orgãos imunes primários, secundários e não imunes de ratos wistar

Abstract

Ectoenzymes play an important role in controlling the signaling developed by adenine nucleotides and nucleosides in countless cells. However, little information is found in the scientific literature about the biochemical characteristics of ectoenzymes that hydrolyze extracellular ATP and ADP in lymphocytes. Thus, this project aimed to carry out the enzymatic characterization of ectoenzymes present in lymphocytes hosting in bone marrow, blood, thymus, cervical and mesenteric lymph nodes, spleen and liver. In a first experiment, we performed the standardization of the tissue lymphocyte isolation technique. In the second experiment, we carried out the enzymatic characterization of the ectonucleotidase activity present in the cells under study. The results found demonstrated that the techniques for the isolation of resident lymphocytes showed high purity in lymphocytes and good viability, essential for the analysis of ectoenzymes. For all tested samples, the enzymatic activity showed linearity as the protein concentration and reaction time increased. The enzymatic hydrolysis occurred at an ideal temperature of 37ºC and in a reaction medium containing a pH value of 8.0. The Chevillard plot confirmed that the hydrolysis of ATP and ADP occurs at the same active site as the enzyme. We confirmed the need for the divalent cations Ca2+ and Mg2+, which showed dose-response up to the concentration of 0.5mM, after this entering in a plateau in the values of enzymatic activity. All of these results are characteristic of the E-NTPDase activity, but to confirm its major action on cells in studies, we performed the analysis of ATDPase inhibitors. We refute the action of other enzymes capable of hydrolyzing extracellular nucleotides, since the inhibitors of these enzymes were not able to inhibit the hydrolysis of ATP and ADP in the samples under analysis. On the other hand, the compounds EDTA (cofactor chelating agent), suramin (non-specific E-NTPDase inhibitor), 20mM sodium azide (capable of inhibiting E-NTPDase activity) and POM-1 (E-NTPDase and E-NPP-1) were able to inhibit enzymatic hydrolysis. It is important to note that we refuted the action of E-NPP-1 in our samples since there was activity in the presence of Ca2+ which is not a cafactor of this ectoenzyme and when adding Zn2+ (cofactor of E-NPP-1) there was no enzymatic activity. When performing the enzymatic kinetics graphs, we found different values of Vmax and km between the lymphocyte isolations. Where the values of Vmax in decreasing order for the lymphocytes isolated from each organ were: liver, spleen, mesenteric lymph nodes, bone marrow, blood, cervical lymph nodes and thymus for both nucleotides. Finally, it is possible to conclude that the hydrolysis of ATP and ADP in the plasma membrane of lymphocytes residing in the primary immune organs (bone marrow and thymus), secondary (spleen and lymph nodes) and non-immune (blood and liver) occurs preferentially by E-NTPDase. Through the enzymatic characterization carried out in this study, we determined a standard value of E-NTPDase activity for each resident lymphocyte. These standardized values are important for future studies that aim to analyze the activity of this ectoenzyme as a marker of lymphocyte activity in pathological processes. In addition, it signals the possible different effects of E-NTPDase modulating agents on the lymphocytes of each tissue.