Churn S B, Taft W C, Billingsley M S, Sankaran B, DeLorenzo R J
Department of Neurology, Medical College of Virginia, Richmond 23298.
J Neurochem. 1992 Oct;59(4):1221-32. doi: 10.1111/j.1471-4159.1992.tb08431.x.
The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.
多功能钙/钙调蛋白依赖性蛋白激酶II（CaM激酶II）的活性最近被证明会受到短暂性全脑缺血的抑制。为了研究缺血诱导的该酶抑制的本质，从对照和缺血沙土鼠的大脑中纯化CaM激酶II，纯化倍数超过1000倍。通过众多参数比较了对照和缺血制剂中CaM激酶II的特性。对纯化的对照和缺血CaM激酶II进行了自磷酸化特性、ATP、镁、钙和钙调蛋白亲和力、免疫反应性及底物识别的动力学分析。缺血诱导了CaM激酶II活性的可重复性抑制，增加任何反应参数的浓度都无法克服这种抑制。缺血CaM激酶II在对钙调蛋白、Ca2+、Mg2+或外源添加底物的亲和力或自磷酸化速率方面与对照酶没有差异。从缺血沙土鼠中分离出的CaM激酶II与针对该酶β亚基的单克隆抗体（免疫球蛋白G3）的免疫反应性降低。此外，通过自磷酸化程度测量时，缺血导致CaM激酶II对ATP的亲和力显著降低。为了进一步表征CaM激酶II对ATP亲和力的降低，使用了共价结合ATP类似物8-叠氮腺苷-5'-[α-32P]三磷酸。与对照酶相比，缺血CaM激酶II中25μM叠氮ATP的共价结合减少了40.4±12.3%（n = 5；配对学生t检验，p < 0.01）。因此，通过蛋白质染色、回收率百分比和钙调蛋白结合，缺血和对照组分的CaM激酶II水平相当，但在免疫反应性和ATP结合方面存在显著差异。这些数据与以下假设一致：缺血诱导了一种翻译后修饰，改变了CaM激酶II中的ATP结合，导致酶活性明显降低。
