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

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

doi:10.1590/1414-431x2020e10850

Abstract

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.

Highlights

Adenosine deaminase (ADA; EC 3.5.4.4), represented in humans by two isoforms, ADA1 and ADA2, catalyzes the conversion of adenosine to inosine [1,2,3]
The results demonstrated that human lymphocyte-rich peripheral blood mononuclear cells (PBMCs) maintained a high viability (Figure 1), even 20 h after separation, which allowed the cells to be used in the subsequent assays, without the need for immediate enzymatic quantification after isolation
The adenosine deamination of human lymphocyte-rich PBMCs isolated from peripheral blood was evaluated as a function of time, protein concentration, and kinetic constant, in order to determine the correct assay conditions

Summary

Introduction

Adenosine deaminase (ADA; EC 3.5.4.4), represented in humans by two isoforms, ADA1 and ADA2, catalyzes the conversion of adenosine to inosine (and deoxyadenosine to deoxyinosine) [1,2,3]. Both human ADA isoforms belong to the zinc-dependent hydrolase family and are strongly inhibited by deoxycoformycin and erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). ADA is present in all cell types, ADA levels differ widely among tissues. The highest ADA levels are found in lymphoid tissues [4], and ADA1 influences lymphocyte function. While certain inherited ADA mutations result in severe immunodeficiency, the physiological role of ADA2 is unknown. ADA2 is found in small amounts in the serum and can be produced by monocytes and macrophages [5]

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