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

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 (K m ) and maximum velocity (V max ), 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 mg of protein, and pH 6.0 to 7.4. The K m and V max values were 0.103 ± 0.051 mM and 0.025 ± 0.001 nmol NH 3 Ámg -1 Ás -1 , respectively. Zinc and erythro-9-(2hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2 0 -deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyterich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

Estimation of Serum Adenosine Deaminase-2 (ADA2) as One of the Non- Invasive Diagnostic Marker of Pleural Effusion in Systemic Lupus Erythromatosus

“…12 ADA has two isoforms, ADA1 and ADA2; ADA1 is found in lymphocytic fluids, while ADA2 is found in plasma and serum. 13 Arthritis and psoriasis cause elevated ADA levels in blood plasma, whereas lung fibrosis causes decreased levels. 14,15 The aim of this study was to evaluate and compare the levels of the ADA2 in blood plasma of SLE patients with and without pleural effusion in this study.…”