Rücker Bárbara, Almeida Manoela E, Libermann Towia A, Zerbini Luiz F, Wink Márcia R, Sarkis João J F
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul - UFRGS, Porto Alegre, RS, Brazil.
Life Sci. 2008 Feb 27;82(9-10):477-86. doi: 10.1016/j.lfs.2007.12.003. Epub 2008 Jan 16.
In this study, we have identified the E-NTPDase family members and ecto-5'-nucleotidase/CD73 in rat heart left ventricle. Moreover, we characterize the biochemical properties and enzyme activities from synaptosomes of the nerve terminal endings of heart left ventricle. We observe divalent cation-dependent enzymes that presented optimum pH of 8.0 for ATP and ADP hydrolysis, and 9.5 for AMP hydrolysis. The apparent K(M) values are 40 microM, 90 microM and 39 microM and apparent V(max) values are 537, 219 and 111 nmol Pi released/min/mg of protein for ATP, ADP and AMP hydrolysis, respectively. Ouabain, orthovanadate, NEM, lanthanum and levamisole do not affect ATP and ADP hydrolysis in rat cardiac synaptosomes. Oligomycin (2 microg/mL) and sodium azide (0.1 mM), both mitochondrial ATPase inhibitors, inhibit only the ATP hydrolysis. High concentrations of sodium azide and gadolinium chloride show an inhibition on both, ATP and ADP hydrolysis. Suramin inhibit more strongly ATP hydrolysis than ADP hydrolysis whereas Evans blue almost abolish both hydrolysis. AMP hydrolysis is not affected by levamisole and tetramisole, whereas 0.1 mM ammonium molybdate practically abolish the ecto-5'-nucleotidase activity. RT-PCR analysis from left ventricle tissue demonstrate different levels of expression of Entpd1 (Cd39), Entpd2 (Cd39L1), Entpd3 (Cd39L3), Entpd5 (Cd39L4) Entpd6, (Cd39L2) and 5'-NT/CD73. By quantitative real-time PCR we identify the Entpd2 as the enzyme with the highest expression in rat left ventricle. Our results contribute to the understanding about the control of the extracellular nucleotide levels in and cardiac system.
在本研究中，我们已鉴定出大鼠心脏左心室中的E-NTPDase家族成员和胞外5'-核苷酸酶/CD73。此外，我们表征了心脏左心室神经末梢突触体的生化特性和酶活性。我们观察到二价阳离子依赖性酶，其对ATP和ADP水解的最佳pH值为8.0，对AMP水解的最佳pH值为9.5。ATP、ADP和AMP水解的表观K(M)值分别为40 microM、90 microM和39 microM，表观V(max)值分别为537、219和111 nmol Pi释放/分钟/毫克蛋白质。哇巴因、原钒酸盐、N-乙基马来酰亚胺、镧和左旋咪唑不影响大鼠心脏突触体中的ATP和ADP水解。线粒体ATP酶抑制剂寡霉素（2 microg/mL）和叠氮化钠（0.1 mM）仅抑制ATP水解。高浓度的叠氮化钠和氯化钆对ATP和ADP水解均有抑制作用。苏拉明对ATP水解的抑制作用比对ADP水解的抑制作用更强，而伊文思蓝几乎完全消除了两者的水解。左旋咪唑和四咪唑不影响AMP水解，而0.1 mM钼酸铵几乎完全消除了胞外5'-核苷酸酶活性。左心室组织的RT-PCR分析显示Entpd1（Cd39）、Entpd2（Cd39L1）、Entpd3（Cd39L3）、Entpd5（Cd39L4）、Entpd6（Cd39L2）和5'-NT/CD73的表达水平不同。通过定量实时PCR，我们确定Entpd2是大鼠左心室中表达最高的酶。我们的结果有助于理解心脏系统中细胞外核苷酸水平的调控。
