Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+-ATPase in E1P and E1ATP states:: a limited proteolysis study
Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+-ATPase in E1P and E1ATP states:: a limited proteolysis study
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DOI：
10.1016/s0014-5793(01)02801-0
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发表时间：
2001-09-07
期刊：
影响因子：
3.5
通讯作者：
Toyoshima, C
中科院分区：
文献类型：
--
作者：
Danko, S;Yamasaki, K;Toyoshima, C
为了研究肌浆网钙-ATPase结构域在不同生理状态下的结构特征，系统地、定量地研究了三种蛋白水解酶(PRTK、V8和Trypsin)对肌浆网钙-ATPase结构域的影响。我们在之前的研究中研究了E-2和E2P之间的差异，并且E2P的特征是对所有三种酶都具有完全抗性(除了分子最顶端的胰酶攻击(T1位点))。本研究对E(1)ATP、E(1)PADP和E1P状态采用了相同的策略。由于这些状态的瞬变性质，它们要么被不可水解的类似物稳定，要么通过调节缓冲条件而占优势。氟化铝(不含ADP)对E1P有稳定作用。所有这些状态都表现出对PRTK和V8的强(E(1)ATP)至完全(E(1)PADP和E1P)抗性，但仅在T2位对胰酶有弱的抗性。由于PRTK和V8主要攻击连接A结构域和跨膜螺旋的环，而胰酶T2位点(Arg(198))位于A结构域的最外面的环上，这些结果导致我们认为A结构域在E11？和E2P。结合前人的研究结果，我们证明通过对三种酶的敏感性可以清楚地区分四种状态，这对于建立结构研究的条件是非常有用的。(C)2001年欧洲生化学会联合会。爱思唯尔科学公司出版。版权所有。
In order to characterize the domain organization of sarcoplasmic reticulum Ca2+-ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E-2 and E2P were examined in our previous study and E2P was characterized by the complete resistance to all three proteases (except for trypsin attack at the very top of the molecule (T1 site)). The same strategies were employed in this study for E(1)ATP, E(1)PADP and E1P states. Because of the transient nature of these states, they were either stabilized by non-hydrolyzable analogues or made predominant by adjusting buffer conditions. Aluminum fluoride (without ADP) was found to stabilize E1P. All these states were characterized by strong (E(1)ATP) to complete (E(1)PADP and E1P) resistance to prtK and to V8 but only weak resistance to trypsin at the T2 site. Because prtK and V8 primarily attack the loops connecting the A domain to the transmembrane helices whereas the trypsin T2 site (Arg(198)) is located on the outermost loop in the A domain, these results lead us to propose that the A domain undergoes a large amount of rotation between Ell? and E2P. Combined with previous results, we demonstrated that four states can be clearly distinguished by the susceptibility to three proteases, which will be very useful for establishing the conditions for structural studies. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.