Dissociation and reconstitution of functional sarcoplasmic reticulum vesicles.
Dissociation and reconstitution of functional sarcoplasmic reticulum vesicles.
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功能性肌浆网囊泡的解离和重建。
DOI：
10.1016/s0021-9258(19)43125-6
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发表时间：
1974
期刊：
影响因子：
--
通讯作者：
S. Fleischer
中科院分区：
文献类型：
--
作者：
G. Meissner;S. Fleischer
功能性肌浆网囊泡的重建已经实现。用3.4mg/ml脱氧胆酸盐溶解浓度为7.0mg/ml蛋白质的肌浆网囊泡。在电子显微镜下观察，溶解的样品是非膜状的。沉降研究表明，它主要分解成蛋白质和磷脂。通过透析去除去污剂导致蛋白质和磷脂的再聚集以形成能够激发Ca 2+积累的膜囊泡。重构是温度依赖性的，在约20°下是最佳的;如果在0-4°下进行透析，则功能不会恢复。有利于复溶的透析缓冲液的组成为0.4mKCl、1.5 mmMg 2 +-1.0 mm亚甲基二胺四乙酸盐和0.1 mmCa 2+，pH 7.25。重组和原始肌浆网囊泡具有相似的磷脂含量和浮力密度。Ca 2+泵蛋白是重组和原始囊泡中的主要蛋白质;然而，分子量约为55，000的另外两种主要蛋白质仅部分反弹。重构的囊泡具有升高的腺苷三磷酸酶活性。在草酸盐存在和不存在下测量的通电Ca 2+吸收能力分别恢复到约50%和25%。
The reconstitution of functional sarcoplasmic reticulum vesicles has been achieved. Sarcoplasmic reticulum vesicles at a concentration of 7.0 mg of protein per ml are solubilized with the use of 3.4 mg per ml of deoxycholate. The solubilized sample is nonmembranous as viewed in the electron microscope. Sedimentation studies indicate it is dissociated largely into protein and phospholipid. Removal of the detergent by dialysis results in the reaggregation of protein and phospholipid to form membranous vesicles which are capable of energized Ca2+accumulation. The reconstitution is temperature dependent being optimal at about 20°; function is not restored if dialysis is carried out at 0–4°. The composition of the dialysis buffer which favors reconstitution is 0.4mKCI, 1.5 mmMg2+-1.0 mmethylenediaminetetraacetate, and 0.1 mmCa2+, pH 7.25. Reconstituted and original sarcoplasmic reticulum vesicles have a similar phospholipid content and buoyant density. The Ca2+pump protein is the major protein in both reconstituted and original vesicles; however, the two other major proteins with a molecular weight of approximately 55,000 are only in part rebound. The reconstituted vesicles have an elevated adenosine triphosphatase activity. Energized Ca2+uptake capacities measured in the presence and absence of oxalate are restored to about 50% and 25%, respectively.