Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP.
Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP.
复制标题
由笼中 ATP 光解引发的酶循环过程中自旋标记 Ca-ATP 酶的微秒旋转动力学。
DOI：
10.1021/bi00098a008
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Thomas,DD
中科院分区：
文献类型：
--
作者：
Lewis,SM;Thomas,DD
明尼苏达大学医学院生化系，明尼苏达州明尼阿波利斯市，55455收到1991年2月6日；修订稿件收到1991年5月24日摘要：我们已经测量了肌浆网(SR)Ca-ATPase的微秒旋转运动作为酶特异性配体的函数，包括那些诱导主动钙转运的配体。我们用马来酰亚胺自旋探针标记了Ca-ATPase，并用饱和转移电子顺磁共振(ST-EPR)检测了旋转动力学。该探针的ST-EPR谱已被证明对微秒级的蛋白质旋转运动敏感，对应于大规模的蛋白质旋转，这些蛋白质旋转应该受到酶的形状、灵活性、蛋白质-蛋白质相互作用(寡聚态)和蛋白质-脂肪相互作用的变化的影响。我们发现，酶-核苷酸和酶-核苷酸/钙状态的运动与没有配体的运动是难以区分的。DMSO的加入确实会降低旋转流动性，DMSO是一种抑制Ca-ATPase活性并稳定磷酸酶的溶剂。然而，在DMSO存在或不存在的情况下，添加磷酸盐来形成磷酸酶并不会显著改变运动。在钙离子转运活跃的稳定状态下，微秒级的旋转迁移率与静止酶的流动性没有什么区别。为了检测在稳态下可能检测不到的迁移率的任何瞬时变化，并提高稳态测量的精度，我们在有钙存在的情况下用激光脉冲光解笼中的三磷酸腺苷，并检测了自旋标记酶的ST-EPR响应，时间分辨率为1 S，没有观察到ST-EPR信号的显著变化，表明在Ca-ATPase循环的瞬时和稳态阶段，有效旋转相关时间的变化不超过10%。虽然蛋白质运动对Ca-ATPase的功能很重要，但这项研究表明，由于酶的形状、灵活性、低聚状态或蛋白质-脂质相互作用的改变而导致的微秒蛋白质旋转迁移率的变化，并不是钙运输循环的一部分。这不排除(1)持续时间小于1 S的瞬时相效应，(2)限速转变后发生的短暂效应，或(3)不影响整体转动迁移率的寡聚单元内的重排。负责将钙转运到肌浆网(SR)1的固有膜蛋白是Ca-ATPase。虽然已经证明Ca-ATPase的正常功能需要微秒的旋转迁移率(Thomas&Hidalgo，1978；Squier等人，1988a，b)，但关于Ca-ATPase运输周期的分子动力学仍然存在一些问题。特别是，有人提出，酶的形状、灵活性或蛋白质-蛋白质相互作用的变化在动力学循环中发挥功能作用[Martonosi等人(1990)审查]。一种可能发挥功能作用的蛋白质-蛋白质相互作用是将Ca-ATPase单体结合成二聚体或四聚体(Martonosi&Beeler，1983)。电子显微镜(Deamer&Baskin，1969；Jilka等人，1975；Scales&Inesi，1976)和荧光能量转移(Vanderkooi等人，1977；Papp等人，1987)都表明存在低聚物。使用冷冻断口电子显微镜结合显微照片的光学衍射分析，Napolitano等人(1983)得出结论，Ca-ATPase在膜中形成二聚体。无论如何，海史密斯和…
Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received February 6, 1991; Revised Manuscript Received May 24, 1991 abstract: We have measured the microsecond rotational motions of the sarcoplasmic reticulum (SR) Ca-ATPase as a function of enzyme-specific ligands, including those that induce active calcium transport. We labeled the Ca-ATPase with a maleimide spin probe and detected rotational dynamics using satura-tion-transfer electron paramagnetic resonance (ST-EPR). This probe’s ST-EPR spectra have been shown to be sensitiveto microsecond protein rotational motion, corresponding to large-scale protein rotations that should be affected bychanges in the enzyme’s shape, flexibility, protein-proteininteractions (oligomeric state), and protein-lipid interactions. We found that the motions of the enzyme-nucleotide and the en-zyme-nucleotide/Ca states are indistinguishable from the motions in the absence of ligands. Rotational mobility does decrease in response to the addition of DMSO, a solvent that inhibits Ca-ATPase activity and stabilizes the phosphoenzyme. However, the addition of phosphate to form phosphoenzyme, in the presence or absence of DMSO, does not change the motions significantly. During the steady state of active calcium transport, the microsecond rotational mobility is indistinguishable from that of the resting enzyme. In order to detect any transient changes in mobility that might not be detectable in the steady state and to improve the precision of steady-state measurements, we photolyzed caged ATP with a laser pulse in the presence of calcium and detected the ST-EPR response from the spin-labeled enzyme, with a time resolution of 1 s. No significant change in the ST-EPR signal was observed, indicating that the effectiverotational correlation time does not change by more than 10% in the transient or steady-state phases of the Ca-ATPase cycle. While protein motion has been shownto be important to the function of the Ca-ATPase, this study indicates that changes in the microsecond protein rotational mobility, which would be caused by changes in the enzyme’s shape, flexibility, oligomeric state, or protein-lipid interactions, do not occur as part of the calcium transport cycle. This does not rule out (1) transient-phase effects lasting less than 1 s,(2) short-lived effects that occur after the rate-limiting transition, or (3) rearrangements within an oligomeric unit that do not affect the overall rotational mobility. e intrinsic membrane protein responsible for the transport of calcium into the sarcoplasmic reticulum (SR) 1 is the Ca-ATPase. While it has been shown that the Ca-ATPase re-quires microsecond rotationalmobility for normal function (Thomas & Hidalgo, 1978; Squier et al., 1988a, b), several questions remain concerning the molecular dynamics of the Ca-ATPase transport cycle. In particular, it has been proposed that changes in the enzyme’s shape, flexibility, or protein-protein interactions play a functional role in the kinetic cycle [reviewed by Martonosi et al.(1990)]. One type of protein-protein interaction that could play a functional role is the oligomeric association of Ca-ATPase monomers into dimers or tetramers (Martonosi & Beeler, 1983). Electron microscopy (Deamer & Baskin, 1969; Jilka et al., 1975; Scales & Inesi, 1976) and fluorescence energy transfer (Vanderkooi et al., 1977; Papp et al., 1987) have both suggested the presence of oligomers. Using freeze-fracture electron microscopy in conjunction with optical diffraction analysis of the micrographs, Napolitano et al.(1983) concluded that the Ca-ATPase forms a dimer in the membrane. How-ever, Highsmith and …
登录
查看更多内容
使用可裂解交联剂可逆控制肌浆网钙 ATP 酶的寡聚相互作用。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Kurobe,Y;Nelson,RW;Ikemoto,N
通讯作者：
Ikemoto,N
影响因子：
3.4
作者：
SCALES, D;INESI, G
通讯作者：
INESI, G
纯化的 (Mg2Ca2) 激活的肌浆网 ATP 酶对磷脂囊泡被动 Ca2 通透性和超微结构的影响。
DOI：
10.1016/s0021-9258(19)40973-3
发表时间：
1975
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. Jilka;A. Martonosi;T. Tillack
通讯作者：
T. Tillack
功能循环过程中可溶性 Ca2+-ATP 酶单体和寡聚体之间的平衡
影响因子：
3.5
作者：
J. Andersen;B. Vilsen
通讯作者：
B. Vilsen
在不添加碱金属盐的情况下，骨骼肌肌浆网 Ca2 依赖性 ATP 水解的反应机制。
DOI：
10.1016/s0021-9258(17)34887-1
发表时间：
1978
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
M. Shigekawa;J. Dougherty
通讯作者：
J. Dougherty