Matejovicová M, Machác S, Lehotský J, Jakus J, Mézesová V
Department of Biochemistry, Jessenius Medical Faculty, Comenius University, Martin, Slovak Republic.
Mol Chem Neuropathol. 1996 Sep;29(1):67-78. doi: 10.1007/BF02815194.
We studied the activity and kinetic parameters of synaptosomal Na, K-ATPase during 15 min of forebrain ischemia and following 60 min of reperfusion produced by reversible common carotid occlusion in Mongolian gerbils. A synaptosomal fraction was obtained by both differential centrifugation of brain tissue homogenate and centrifugation of crude mitochondrial fraction at a discontinual sucrose density gradient. We found two components of ATP concentration dependence of ATP hydrolysis that represent two types of ATP-binding sites: high affinity and low affinity. Neither ischemia nor reperfusion affected kinetic parameters of a high-affinity site. However, low-affinity site parameters were affected by both ischemia and ischemia followed by reperfusion. Maximal velocity (Vmax) decreased by 43 and 42% after ischemia and after ischemia/reperfusion, respectively. The apparent Km for ATP decreased by 52% after ischemia and by 47% after ischemia/reperfusion. The apparent affinities for K+ and Na+ were determined from the ATP hydrolysis rate as a function of Na+ and K+ concentrations. We found the half-maximal activation constant for K+ (KaK+) increased by 60% after ischemia and by 146% after ischemia/reperfusion. On the other hand, we found that KaNa+ decreased significantly after ischemia/reperfusion (16%). We concluded that it is the dephosphorylation step of the ATPase reaction cycle that is primarily affected by both ischemia and ischemia/reperfusion. This might be caused by alteration of the protein molecule and/or its surroundings subsequent to ischemia.
我们研究了蒙古沙鼠前脑缺血15分钟及随后由可逆性颈总动脉闭塞产生的60分钟再灌注期间突触体钠钾ATP酶的活性和动力学参数。通过脑组织匀浆的差速离心和粗线粒体部分在间断蔗糖密度梯度下的离心获得突触体部分。我们发现ATP水解的ATP浓度依赖性有两个成分，代表两种类型的ATP结合位点：高亲和力和低亲和力。缺血和再灌注均未影响高亲和力位点的动力学参数。然而，低亲和力位点参数受缺血以及缺血后再灌注的影响。缺血后和缺血/再灌注后最大速度（Vmax）分别降低了43%和42%。ATP的表观Km在缺血后降低了52%，在缺血/再灌注后降低了47%。根据ATP水解速率作为Na+和K+浓度的函数来确定对K+和Na+的表观亲和力。我们发现缺血后K+的半最大激活常数（KaK+）增加了60%，缺血/再灌注后增加了146%。另一方面，我们发现缺血/再灌注后KaNa+显著降低（16%）。我们得出结论，ATP酶反应循环的去磷酸化步骤主要受缺血和缺血/再灌注的影响。这可能是缺血后蛋白质分子和/或其周围环境改变所致。
