Shackelford D A, Yeh R Y, Zivin J A
Department of Neurosciences, University of California, San Diego, La Jolla.
J Neurochem. 1993 Aug;61(2):738-47. doi: 10.1111/j.1471-4159.1993.tb02180.x.
Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II alpha and beta subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the alpha and beta subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (< or = 25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.
兔可逆性脊髓缺血导致钙调蛋白依赖性蛋白激酶II（CaM激酶II）活性迅速丧失，该活性通过磷酸掺入外源底物来测定。在该模型中，在不可逆性截瘫（25分钟）前15分钟的缺血后，脊髓匀浆胞质部分约70%的活性丧失。酶活性的丧失与原位可复性自磷酸化活性的丧失以及通过免疫印迹检测到的胞质中CaM激酶IIα和β亚基的丧失相关。微粒体部分的CaM激酶II活性也降低，但α和β亚基的蛋白质水平增加。因此，缺血导致CaM激酶II失活以及该酶的顺序性或同时性亚细胞重新分布。然而，固定在膜上的CaM激酶亚基的原位变性和复性部分逆转了微粒体部分酶的明显失活。短暂（≤25分钟）缺血后再灌注可恢复CaM激酶II活性，但导致不可逆性截瘫的较长时间（60分钟）缺血后则不能恢复。缺血诱导的CaM激酶II失活，该激酶可磷酸化调节许多细胞过程的蛋白质，可能在导致延迟性神经元细胞死亡的一系列事件中起重要作用。
