Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid
Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid
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使用单宁酸揭示肌浆网的膜不对称性和增强的超微结构细节
DOI：
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发表时间：
1978
影响因子：
7.8
通讯作者：
S. Fleischer
中科院分区：
文献类型：
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作者：
A. Saito;C. T. Wang;S. Fleischer
用加入1%单宁酸的戊二醛固定纯化的肌浆网(SR)膜小泡，在薄片上可见新的超微结构。膜的三层外观高度不对称；外层电子不透明层(70A)比内层(20A)略宽。这种不对称性并不意味着单宁酸缺乏穿透，因为：(A)用1 mM EDTA，pH 8.5使SR囊泡具有类似的不对称性；(B)用胰酶处理SR导致蛋白质含量的进行性损失和外层厚度的减少，直到极限三层出现对称的外观；(C)在相同的肌肉切片内，SR膜看起来高度不对称，而肌膜具有更对称的外观；(D)重组的SR囊泡具有对称的外观，内外层等宽(约70A)；冷冻断口和负染电子显微镜证实了其对称结构。等密度沉淀法获得的重泡和轻泡具有相同的膜不对称外观，主要不同之处在于重泡的内部内容物主要由钙结合蛋白组成。SR膜的不对称性主要与膜的主要成分(90%的蛋白质)钙泵蛋白的单向排列有关。肌浆网膜的不对称性现在第一次可以在肌肉的薄片上观察到。
Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.