Synaptosomal Na, K-ATPase during forebrain ischemia in Mongolian gerbils

Abstract

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.