Department of Molecular Biology and Endocrinology, Institute of Nuclear Sciences VINČA, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia.
Mol Cell Biochem. 2012 Dec;371(1-2):199-208. doi: 10.1007/s11010-012-1436-2. Epub 2012 Sep 7.
Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K (m) values were 506.4 ± 62.1 and 638.8 ± 31.3 μM, with a calculated V (max) (app) of 3,973.0 ± 279.5 and 2,853.9 ± 79.8 nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5 mM). According to similar apparent K(m) values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology.
细胞外核苷酸影响女性生殖功能、受精和妊娠。本研究旨在研究来自 Wistar 白化大鼠子宫肌细胞膜中 ATP 和 ADP 水解的生化特性，并鉴定 E-NTPDases。表观 K(m)值分别为 506.4±62.1 和 638.8±31.3 μM，计算出的 V(max)(app)分别为 3973.0±279.5 和 2853.9±79.8 nmol/min/mg 用于 ATP 和 ADP。此处描述的酶活性具有 NTPDases 的共同特性：二价阳离子依赖性；两种底物的碱性 pH 最佳；对一些经典的 ATPase 抑制剂（哇巴因、寡霉素、茶碱、左旋咪唑）不敏感，对苏拉明和高浓度叠氮化钠（5 mM）有明显抑制作用。根据两种底物的相似表观 K(m)值、ATP/ADP 水解比和 Chevillard 竞争图，NTPDase1 是子宫肌细胞膜中占主导地位的 ATP/ADP 水解酶。RT-PCR 分析显示，在大鼠子宫中表达三种外核苷酸三磷酸二磷酸水解酶家族（NTPDase 1、2 和 8）的成员。这些发现可能进一步阐明 NTPDases 和 ATP 在生殖生理学中的作用。
