Biochemical characterization of adenosine deaminase (CD26; EC 3.5.4.4) activity in human lymphocyte-rich peripheral blood mononuclear cells.

L R Costa, A K Y de Souza, J N Scholl, F Figueiró, A M O Battastini, J A Dos Santos Jaques, F F Zanoelo

Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 2021

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The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2'-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

Citation

L R Costa, A K Y de Souza, J N Scholl, F Figueiró, A M O Battastini, J A Dos Santos Jaques, F F Zanoelo. Biochemical characterization of adenosine deaminase (CD26; EC 3.5.4.4) activity in human lymphocyte-rich peripheral blood mononuclear cells. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas. 2021;54(8):e10850