Romero Ana, Cumplido-Laso Guadalupe, Fernández Ascensión, Moreno Javier, Canales José, Ferreira Rui, López-Gómez Juan, Ribeiro João Meireles, Costas María Jesús, Cameselle José Carlos
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Medicina y Ciencias de la Salud, Universidad de Extremadura, 06006, Badajoz, Spain.
Departamento de Bioquímica y Biología Molecular y Genética, Facultad de Ciencias, Universidad de Extremadura, 06006, Badajoz, Spain.
Amino Acids. 2024 Dec 6;57(1):1. doi: 10.1007/s00726-024-03431-4.
Nucleotide-pyrophosphatases/phosphodiesterases (NPP/PDE) are membrane or secreted Zn-metallohydrolases of nucleoside-5´-monophosphate derivatives. They hydrolyze, for instance, ATP and 4-nitrophenyl-dTMP, and belong to the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family that contains seven members (ENPP1-ENPP7). Earlier we had shown that an NPP/PDE activity solubilized and partially purified from rat liver membranes is inactivated by EDTA in a time-dependent fashion, an effect enhanced by glycine and blocked by the 4-nitrophenyl-dTMP. Here, we extended this observation to other free amino acids. Activity assays started after different incubation lengths with EDTA provided first-order, apparent inactivation constants (k). With the exception of cysteine (a strong inhibitor) and histidine (itself evoking a time-dependent inactivation), free amino acids themselves did not affect activity but increased k. The results are compatible with a conformational change of NPP/PDE evoked by interaction with free amino acids. The enzyme preparation was analyzed to identify what ENPP family members were present. First, the hydrolytic activity on 2´,3´-cGAMP was assayed because until very recently ENPP1 was the only mammalian enzyme known to display it. 2´,3´-cGAMP hydrolase activity was clearly detected, but mass spectrometry data obtained by LC-MS/MS gave evidence that only rat Enpp3, Enpp4 and Enpp5 were present with low abundance. This finding coincided in time with a recent publication claiming that mouse Enpp3 hydrolyzes 2´,3´-cGAMP, and that Enpp1 and Enpp3 account for all the 2´,3´-cGAMP hydrolase activity in mice. So, our results are confirmatory of Enpp3 activity towards 2´,3´-cGAMP. Finally, the effect of amino acids could be relevant to NPP/PDE actions dependent on protein-protein interactions, like the known insulin-related effects of ENPP1 and possibly ENPP3.
核苷酸焦磷酸酶/磷酸二酯酶（NPP/PDE）是核苷5'-单磷酸衍生物的膜结合或分泌型锌金属水解酶。例如，它们能水解ATP和4-硝基苯基-dTMP，属于胞外核苷酸焦磷酸酶/磷酸二酯酶（ENPP）家族，该家族包含七个成员（ENPP1 - ENPP7）。我们之前已经表明，从大鼠肝细胞膜中溶解并部分纯化得到的NPP/PDE活性会被EDTA以时间依赖性方式失活，甘氨酸可增强这种效应，而4-硝基苯基-dTMP可阻断这种效应。在此，我们将这一观察结果扩展到其他游离氨基酸。在与EDTA孵育不同时长后开始进行活性测定，得到了一级表观失活常数（k）。除了半胱氨酸（一种强抑制剂）和组氨酸（其本身会引起时间依赖性失活）外，游离氨基酸本身并不影响活性，但会增加k。这些结果与游离氨基酸相互作用引起的NPP/PDE构象变化相符。对酶制剂进行分析以确定存在哪些ENPP家族成员。首先，检测了对2´,3´-cGAMP的水解活性，因为直到最近ENPP1还是已知唯一具有该活性的哺乳动物酶。明显检测到了2´,3´-cGAMP水解酶活性，但通过液相色谱-串联质谱（LC-MS/MS）获得的质谱数据表明，仅存在低丰度的大鼠Enpp3、Enpp4和Enpp5。这一发现与最近一篇声称小鼠Enpp3能水解2´,3´-cGAMP且Enpp1和Enpp3构成小鼠所有2´,3´-cGAMP水解酶活性的论文同时出现。所以，我们的结果证实了Enpp3对2´,3´-cGAMP的活性。最后，氨基酸的作用可能与依赖蛋白质-蛋白质相互作用的NPP/PDE作用相关，比如ENPP1以及可能的ENPP3已知的与胰岛素相关的作用。
