Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity.
Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity.
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DOI：
10.1016/s0021-9258(18)67200-x
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发表时间：
1986-09
期刊：
影响因子：
--
通讯作者：
G. Demartino;C. A. Huff;D. E. Croall
中科院分区：
文献类型：
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作者：
G. Demartino;C. A. Huff;D. E. Croall
来自牛心脏的钙依赖性蛋白酶 II (CDP-II) 是一种异二聚体，亚基分子量为 80,000 和 26,000。先前的研究表明，该蛋白酶需要 350 microM Ca2+ 才能达到最大活性的一半，并且大亚基同时包含该酶的催化和 Ca2+ 结合功能。小亚基的功能尚不清楚。我们研究了在存在和不存在底物蛋白的情况下 Ca2+ 对 CDP-II 的结构和催化特性的影响。当在没有底物的情况下与 Ca2+ 一起孵育时，CDP-II 会经历一系列自蛋白水解裂解，依次将小亚基的分子量从 26,000 降低至 24,000、22,000 至 17,000。在此期间，80 kDa 亚基没有可检测到的变化，该亚基仍与自溶小亚基相关。自蛋白水解速率取决于温度和 Ca2+ 浓度（约 600 microM Ca2+ 时的半最大速率）。第一次裂解似乎是单分子的，因为其速率不受 CDP-II 浓度或外源蛋白质底物存在的影响。随后的裂解导致 80-kDa/17-kDa 异二聚体的形成，并且似乎是通过双分子反应发生的；这些反应的速率通过降低 CDP-II 浓度和蛋白质底物的存在而减慢。小亚基的自蛋白水解具有两种不同的功能后果，每种后果都与不同形式的自溶蛋白酶相关。我们的结果表明，80-kDa/26-kDa 形式的 CDP-II 代表一种无活性的酶原，并且 26-kDa 亚基的初始 Ca2+ 依赖性裂解导致蛋白酶激活。激活的酶水解蛋白质底物，Ca2+ 浓度要求为 350 µM，达到半最大速率。进一步的自蛋白水解导致 80-kDa/17-kDa 异二聚体的形成，可将蛋白酶活性所需的 Ca2+ 浓度降低 25 倍。因此，这些结果为小亚基在调节 CDP-II 活性中的特定作用提供了证据。
Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM Ca2+ for half-maximal activity and that the large subunit contains both the catalytic and Ca2+ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca2+ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our results indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca2+-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca2+ concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the Ca2+ concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity.