Dias Dhébora Albuquerque, de Barros Penteado Bruna, Dos Santos Lucas Derbocio, Dos Santos Pedro Mendes, Arruda Carla Cardozo Pinto, Schetinger Maria Rosa Chitolina, Leal Daniela Bitencourt Rosa, Dos Santos Jaques Jeandre Augusto
Laboratório de Bioquímica Geral e de Microrganismos, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
Programa de Pós-Graduação em Farmácia, Faculdade de Farmácia Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
Cell Biochem Funct. 2017 Oct;35(7):358-363. doi: 10.1002/cbf.3281. Epub 2017 Sep 4.
This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E-NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca . A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E-NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.
本研究旨在表征BALB/c小鼠腹腔细胞中外核苷酸三磷酸二磷酸水解酶（E-NTPDase；EC 3.6.1.5）的活性。E-NTPDase在钙离子（1.5mM）和镁离子（1.5mM）同时存在的情况下被激活。然而，在钙离子存在时活性更高。pH 8.5和37°C是催化的最佳条件。三磷酸腺苷（ATP）和二磷酸腺苷（ADP）水解的表观Km值分别为0.51mM和0.66mM。ATP酶和ADP酶活性的Vmax值分别为136.4和120.8 nmol无机磷/分钟/毫克蛋白质。在叠氮化钠（20mM，ATP：P <.05；ADP：P <.001）、氟化钠（20mM；ATP和ADP：P <.001）和苏拉明（0.3mM；ATP：P <.01；ADP：P <.05）存在时，核苷酸水解受到抑制，这是NTPDase抑制的已知特征。尽管所有测试的二磷酸和三磷酸核苷酸都被水解，但当腺嘌呤核苷酸用作底物时，酶活性增加。最后，我们强调，了解小鼠腹腔细胞中E-NTPDase催化的生化特性对于正确测定其活性以及全面了解健康和患病细胞中的免疫反应情况是不可或缺的。
