Romanowska Małgorzata, Ostrowska Marta, Komoszyński Michał A
Department of Biochemistry, Faculty of Biology and Earth Sciences, Nicolaus Copernicus University, 9 Gagarina St., 87-100 Toruń, Poland.
Brain Res. 2007 Jul 2;1156:1-8. doi: 10.1016/j.brainres.2007.04.037. Epub 2007 Apr 20.
We have purified and investigated the role of adenosine ecto-deaminase (ecto-ADA) in porcine brain synaptic membranes and found a low activity of ecto-ADA in synaptic preparations from the cerebral cortex, hippocampus, striatum and medulla oblongata in the presence of purine transport inhibitors (NBTI, dipyridamole and papaverine). The purification procedure with affinity chromatography on epoxy-Toyopearl gel/purine riboside column as a crucial step of purification allowed a 214-fold purification of synaptic ecto-ADA with a yield of 30%. Gel filtration chromatography revealed a molecular mass estimated at 42.4+/-3.9 kDa. The enzyme had a broad optimum pH and was not affected by mono- and divalent cations. Ecto-ADA revealed a low affinity to adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) (K(M)=286.30+/-40.38 microM and 287.14+/-46.50 microM, respectively). We compared the affinity of ecto-ADA to the substrates with the physiological and pathological concentrations of the extracellular Ado in brains that do not exceed a low micromolar range even during ischemia and hypoxia, and with the affinity of adenosine receptors to Ado not exceeding a low nanomolar (A(1) and A(2A) receptors) or low micromolar (A(2B) and A(3)) range. Taken together, our data suggest that the role of synaptic ecto-ADA in the regulation of the ecto-Ado level in the brain and in the termination of adenosine receptor signaling is questionable. The porcine brain synapses must have other mechanisms for the ecto-Ado removal from the synaptic cleft and synaptic ecto-ADA may also play an extra-enzymatic role in cell adhesion and non-enzymatic regulation of adenosine receptor activity.
我们已纯化并研究了腺苷胞外脱氨酶（ecto - ADA）在猪脑突触膜中的作用，发现在存在嘌呤转运抑制剂（NBTI、双嘧达莫和罂粟碱）的情况下，大脑皮层、海马体、纹状体和延髓的突触制剂中ecto - ADA的活性较低。以环氧 - 东曹凝胶/嘌呤核苷柱上的亲和色谱法作为纯化的关键步骤，该纯化程序使突触ecto - ADA得到了214倍的纯化，产率为30%。凝胶过滤色谱显示分子量估计为42.4±3.9 kDa。该酶具有较宽的最适pH值，且不受单价和二价阳离子的影响。ecto - ADA对腺苷（Ado）和2'-脱氧腺苷（2'-dAdo）的亲和力较低（K(M)分别为286.30±40.38 μM和287.14±46.50 μM）。我们将ecto - ADA对底物的亲和力与大脑中细胞外Ado的生理和病理浓度进行了比较，即使在缺血和缺氧期间，大脑中细胞外Ado的浓度也不超过低微摩尔范围，并且将其与腺苷受体对Ado的亲和力进行了比较，腺苷受体对Ado的亲和力不超过低纳摩尔（A(1)和A(2A)受体）或低微摩尔（A(2B)和A(3)）范围。综上所述，我们的数据表明，突触ecto - ADA在调节大脑中胞外Ado水平以及终止腺苷受体信号传导方面的作用值得怀疑。猪脑突触必定具有其他从突触间隙清除胞外Ado的机制，并且突触ecto - ADA在细胞黏附和腺苷受体活性的非酶调节中可能也发挥着酶外作用。
