Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase.
Effect of urea on the partial reactions and crystallization pattern of sarcoplasmic reticulum adenosine triphosphatase.
复制标题
尿素对肌浆网三磷酸腺苷酶部分反应和结晶模式的影响。
DOI：
10.1016/0003-9861(88)90373-6
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发表时间：
1988
影响因子：
3.9
通讯作者：
Wade,JB
中科院分区：
文献类型：
--
作者：
Jorge-Garcia,I;Bigelow,DJ;Inesi,G;Wade,JB
稳态ATP酶活性，钙结合，形成磷酸化的酶中间体与ATP的存在下的Ca ~（2+），或与Pi，在Ca ~（2+）的情况下，和协会的ATP酶分子成二维晶体，进行了研究，使用囊泡片段的肌浆网。囊泡暴露于浓度增加的尿素，以产生逐步扰动的蛋白质结构和测试的影响，这种扰动的部分反应和结晶模式的肌浆网ATP酶。发现低浓度的尿素对Pi结合和酶磷酸化产生特异性抑制作用（但对ATP不产生抑制作用）。中间浓度的尿素降低钙结合亲和力和协同性，而酶被ATP磷酸化并形成二聚体阵列的能力得以保留。这些观察结果表明，肌浆网ATP酶是敏感的物理扰动产生特定的和可逆的变化，在thePi和钙结合域。这些变化干扰酶的周转，表明构象的影响有关的绑定和解离ofpi和钙紧密耦合到催化和能量转导。较高浓度的尿素产生不可逆的变性，伴随着钙结合的完全抑制，酶与ATP的磷酸化，以及ATP酶链在二维晶体中的缔合。在这些条件下，蛋白质解折叠表现为内在色氨酸残基和共价结合探针的荧光急剧减少。这些观察结果表明，二聚体缔合和形成二维晶体的趋势对应于酶的基本性质，其与其天然结构相关，并且其特征可能在配体存在下和/或在催化循环期间发生变化。另一方面，十钒酸盐诱导的结晶模式不能解释的ATP酶二聚化的催化循环的中间状态之一的机械关系。
Steady-state ATPase activity, calcium binding, formation of phosphorylated enzyme intermediate with ATP in the presence of Ca2+, or withPi, in the absence of Ca2+, and association of ATPase molecules into bidimensional crystals, were studied using vesicular fragments of sarcoplasmic reticulum. The vesicles were exposed to increasing concentrations of urea in order to produce stepwise perturbations of protein structure and to test the effect of such perturbations on the partial reactions and crystallization pattern of sarcoplasmic reticulum ATPase. It was found that low concentrations of urea produce specific inhibition ofPibinding and enzyme phosphorylation withPi(but not with ATP). Intermediate concentrations of urea reduce calcium binding affinity and cooperativity, while the ability of the enzyme to be phosphorylated with ATP and to form dimeric arrays is retained. These observations demonstrate that the sarcoplasmic reticulum ATPase is sensitive to physical perturbations producing specific and reversible changes in thePiand calcium binding domains. These changes interfere with enzyme turnover, indicating that conformational effects related to binding and dissociation ofpiand calcium are tightly coupled to catalysis and energy transduction. Higher concentrations of urea produce irreversible denaturation, accompanied by total inhibition of calcium binding, enzyme phosphorylation with ATP, and association of ATPase chains in bidimensional crystals. Under these conditions, protein unfolding is manifested by a sharp reduction in the fluorescence of intrinsic tryptophan residues and of a covalently bound probe. These observations suggest that dimeric association and a tendency to form bidimensional crystals correspond to a basic property of the enzyme, which is linked to its native structure and whose character may change in the presence of ligands and/or during the catalytic cycle. On the other hand, the decavanadate-induced crystallization pattern cannot be interpreted in terms of a mechanistic relationship of ATPase dimerization with one of the intermediate states of the catalytic cycle.