Characterization of E ‐NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes

The activity of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E‐NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E‐NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca2+. In addition, in the presence of specific E‐NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E‐ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03μM and 214.40 ± 0.06μM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 ƞmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E‐NTPDase. The higher E‐NTPDase activity observed in HL may be attributed to the important physiological ...
Source: Cell Biochemistry and Function - Category: Biochemistry Authors: Tags: RESEARCH ARTICLE Source Type: research