Health and Medical Institute, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-4 Umezono, Tsukuba 305-8568, Japan.
Institute for Excellence in Higher Education, Tohoku University, 41 Kawauchi, Aoba-ku, Sendai 980-8576, Japan.
Int J Mol Sci. 2023 Apr 11;24(8):7080. doi: 10.3390/ijms24087080.
[Ca]-dependent crystallization of the Ca-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca and almost disappeared at ≥18 µM Ca, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.
从扇贝横纹肌中分离出的肌浆网（SR）囊泡中的 Ca-ATPase 分子在 Ca 依赖性结晶作用下，在没有 ATP 的情况下使囊泡伸长，而 ATP 稳定了晶体。在这里，为了确定在存在 ATP 的情况下囊泡伸长对 Ca 的依赖性，使用负染色电子显微镜对处于不同 Ca 环境中的 SR 囊泡进行成像。所获得的图像揭示了以下现象。（i）在 ≤1.4 µM Ca 时出现含有晶体的伸长囊泡，而在 ≥18 µM Ca 时几乎消失，此时 ATPase 活性达到最大值。（ii）在 ≥18 µM Ca 时，几乎所有的 SR 囊泡都呈圆形，并且被紧密聚集的 ATPase 晶体斑块覆盖。（iii）在电子显微镜网格上干燥的圆形囊泡偶尔会出现裂缝，可能是因为表面张力压碎了固态的三维球体。（iv）Ca 依赖性 ATPase 结晶快速（<1 分钟）且可逆。这些数据提示了一个假说，即 SR 囊泡在钙敏感的 ATPase 网络/内骨骼的帮助下自主伸长或收缩，并且 ATPase 结晶可能调节 SR 结构的物理性质，包括控制肌肉收缩的ryanodine 受体。
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