Conformational changes in the vicinity of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle.
Conformational changes in the vicinity of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle.
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在整个催化循环中，附着在肌浆网 Ca2-ATP 酶特定硫醇上的 N-碘乙酰基-N-(5-磺基-1-萘基)乙二胺附近的构象变化。
DOI：
10.1016/s0021-9258(18)68980-x
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
T. Kanazawa
中科院分区：
文献类型：
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作者：
M. Obara;H. Suzuki;T. Kanazawa
在先前的实验中（Suzuki，H.，Obara，M.，Kuwayama，H.，和金泽、T.（1987）J.Biol.Chem.262，15448-15456），用N-碘乙酰基-N’-（5-磺基-1-萘基）乙二胺标记肌浆网囊泡的Ca 2 +-ATP酶，而不损失催化活性。主要标记位点为Cys 674。在K+存在下，ATP与Ca ~（2+）激活的酶的催化位点结合时，发生大的双相荧光下降。目前的结果表明，这种荧光下降是双相的K+的情况下。这种下降的第一和快速阶段占荧光下降的大部分。这个相反映了酶的构象变化。ATP复合物。第二和缓慢的阶段，是远远小于第一阶段，符合磷酸酶（EP）异构化从ADP敏感的形式ADP不敏感的形式。当ADP不敏感EP的积累被K+抑制或当EP异构化被N-乙基马来酰亚胺处理阻止时，该阶段消失。这些结果表明，该相反映了EP异构化后的构象变化。当游离Ca 2+在由ATP形成EP后被螯合时，荧光强度恢复到没有Ca 2+的初始水平。这种恢复与EP分解一致。这表明荧光恢复反映了ADP不敏感EP水解后的构象变化。这种可能性得到了Pi诱导的荧光下降和Pi的EP形成的同时发生的支持。结果表明，ATP结合后的荧光下降是整个催化循环中荧光变化的主要原因。
In the previous experiment (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456), the Ca2+-ATPase of sarcoplasmic reticulum vesicles was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The main labeled site was Cys674. A large monophasic fluorescence drop occurred upon ATP binding to the catalytic site of the Ca2+-activated enzyme in the presence of K+. The present results show that this fluorescence drop is biphasic in the absence of K+. The first and rapid phase of this drop accounts for most of the fluorescence drop. This phase reflects a conformational change in the enzyme.ATP complex. The second and slow phase, being much smaller than the first phase, coincides with phosphoenzyme (EP) isomerization from the ADP-sensitive form to the ADP-insensitive form. This phase disappears when accumulation of ADP-insensitive EP is inhibited by K+ or when EP isomerization is prevented by the N-ethylmaleimide treatment. These results show that this phase reflects a conformational change upon EP isomerization. When free Ca2+ is chelated after EP formation from ATP, the fluorescence intensity is restored to the initial level without Ca2+. This restoration coincides with EP decomposition. This suggests that the fluorescence restoration reflects a conformational change upon hydrolysis of ADP-insensitive EP. This probability is supported by the concurrent occurrence of the Pi-induced fluorescence drop and EP formation from Pi. The results demonstrate that the fluorescence drop upon ATP binding is predominant in the fluorescence change throughout the catalytic cycle.