Characterization of adenosine deaminase (ADA) in Hemolymph of Biomphalaria glabrata

Abstract

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 µmols.L-1. Highest activity was found at 37ºC. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 µL of pure hemolymph per assay, pH 6.8, and 37ºC. This enzyme showed activities of 834 ± 67 µmol.min-1.L-1 (25ºC) and 2029 ± 74 µmol.min-1.L-1 (37ºC), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43ºC or 70% at 50ºC for 15 minutes. The ADA lost from 26 to 78% of its activity when hemolymph was pre-incubated at 50ºC for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.

Adenosine deaminase from camel tick Hyalomma dromedarii: purification and characterization

“…ADAII was stable up to 40°C and lost 80% of its activity after incubation at 70°C for 15 min. Adenosine deaminase from the hemolymph of Biomphalaris glabrata lost 78% of its activity when preincubated at 50°C for 15 min (Vale 2005). Adenosine deaminase from camel skeletal muscles fully inactivated after 20 min when incubated at 70°C (Alrokayan 2002).…”

“…This enzyme is of special interest for its role in regulation of cellular growth and differentiation and it is an important signaling molecule for many cellular events (Hershfield and Mitchell 1995;Vale et al 2005). The physiological roles of adenosine deaminase are directly related to the control of adenosine concentrations in intra-and extra-cellular space (Vale et al 2005).…”

“…This enzyme is of special interest for its role in regulation of cellular growth and differentiation and it is an important signaling molecule for many cellular events (Hershfield and Mitchell 1995;Vale et al 2005). The physiological roles of adenosine deaminase are directly related to the control of adenosine concentrations in intra-and extra-cellular space (Vale et al 2005). Adenosine deaminase converts the vasodilatory, antiplatelet and immunosuppressive molecule adenosine (Webster 1984;Charlab et al 2000) into inosine, which also has multiple anti-inflammatory effects (Hasko et al 2000).…”

“…Adenosine deaminase converts the vasodilatory, antiplatelet and immunosuppressive molecule adenosine (Webster 1984;Charlab et al 2000) into inosine, which also has multiple anti-inflammatory effects (Hasko et al 2000). This enzyme has been studied in invertebrates such as marine mollusks (Chen et al 2000), sand fly Lutzomyia longipalpis (Charlab et al 2000), mosquito Culex pipiens (Ribeiro et al 2001) and Biomphalaria glabrata (Vale et al 2005).…”

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

“…The quantification of ADA activity has been previously performed in sheep lymphoid cells during antibody production after antigenic stimulation (10), peripheral blood cells of patients with hematological disorders, such as lymphoid leukemia and myeloma (11), serum from patients with different liver disorders (12), serum from patients with acquired immunodeficiency virus (HIV) infection ( 13), synovial fluid from patients with rheumatoid arthritis (14), serum from patients with tuberculous meningitis (15), serum from patients with chronic lymphoid leukemia (16), and other sources. ADA activity has also been characterized in leukemic cells (17), normal epidermal and carcinoma cells (18), mouse intestines (19), human and chicken livers (20), mollusk (Biomphalaria glabrata) hemolymph (21), and zebrafish brain (22). Although Giusti (23) and Guisti et al (24) have characterized the enzymatic activity of ADA in human serum, no study has characterized ADA activity in isolated blood lymphocytes in detail, despite that many of the aforementioned studies quantified its activity in these specific cells.…”