Intact Vesicles of Canine Cardiac Sarcolemma: Evidence from Vectorial Properties of Na+,K+-ATPase
Intact Vesicles of Canine Cardiac Sarcolemma: Evidence from Vectorial Properties of Na+,K+-ATPase
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犬心脏肌膜的完整囊泡：来自 Na ,K -ATP 酶矢量特性的证据
DOI：
10.1161/01.res.39.4.586
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发表时间：
1976
影响因子：
20.1
通讯作者：
A. M. Watanabe
中科院分区：
文献类型：
--
作者：
H. R. Besch;L. Jones;A. M. Watanabe
大多数生物膜在功能上是不对称的。为了研究心脏跨肌膜离子流的生化控制，使用分离的肌膜囊泡将具有明显的优势，该囊泡保留了完整肌膜的低被动渗透性特征，因为在这样的囊泡中，膜应表现出其关于酶活性的正常不对称特征。本研究的目的是尝试鉴定心脏微粒体（膜囊泡）制剂中的此类囊泡。我们在 Mg2+ 存在下使用 ATP 或对硝基苯磷酸 (pNPP) 作为底物，研究了 Na+、K+= ATP 酶的 Na+ 和 K+ 激活及其相关的 K+-磷酸酶活性。单独 K+ 的最佳浓度 (10 mM) 可刺激对硝基苯磷酸酶 (p NPPase) 活性 1.8 倍，并且超过 80% 的增加可被哇巴因抑制。最佳 Na 加 K' 浓度（分别为 100 mM 和 10 DIM）可将 ATP 水解速率刺激 2 倍，但只有 11 ± 1.1% 的增加活性对哇巴因敏感。用十二烷基硫酸钠 (SDS) (0.3 mg/ml) 进行最佳预处理，使两种活性对哇巴因的抑制完全敏感，并将基础 Mg2+-ATPase 活性降低 70-90%。在 SDS 中预孵育后，K+ 刺激的 pNPPase 活性加倍，但在这些条件下，Na+ 加 K+ 刺激的 ATPase 活性下降了 50%。通过与脱氧胆酸盐 (DOC) 预孵育产生类似的表观激活模式，不同之处在于基础 Mg1+ 依赖性活性能够抵抗这种去污剂的破坏。对离子和底物的激活以及哇巴因的抑制的增量响应与以下假设一致：分离的制剂中存在渗透性完整的肌膜囊泡，并且去污剂活化使囊泡对离子、底物和哇巴因具有高度渗透性。
Most biological membranes are functionally asymmetric. To study biochemical control of cardiac transsarcolemmal ion fluxes, it would be of obvious advantage to use isolated vesicles of sarcolemma which retains the low passive permeability characteristics of intact sarcolemma because in such vesicles the membrane should exhibit its normal asymmetric character with respect to enzymic activities. The purpose of this investigation was to attempt to identify such vesicles in a cardiac microsomal (membrane vesicular) preparation. We studied activation by Na+ and K+ of Na+,K+= ATPase and its associated K+-phosphatase activities, using as substrates ATP or p-nitrophenytphosphate (pNPP) in the presence of Mg2+. Optimal concentrations of K+ alone (10 mM) stimulated p-nitrophenylphosphatase (p NPPase) activity 1.8-fold, and over 80% of the increase could be inhibited by ouabain. Optimal Na plus K' concentrations (100 mM and 10 DIM, respectively) stimulated the rate of ATP hydrolysis 2-fold, but only 11 ± 1.1% of the increased activity was ouabain-sensitive. Optimal pretreatment with sodium dodecyl sulfate (SDS) (0.3 mg/ml) rendered both activities completely sensitive to inhibition by ouabain and reduced the basal Mg2+-ATPase activity by 70–90%. The K+-stimulated pNPPase activity doubled after preincubation in SDS, but the ATPase activity stimulated by Na+ plus K+ fell by 50% under these conditions. A similar pattern of apparent activation was produced by preincubation with deoxycholate (DOC), except that basal Mg1+-dependent activities were resistant to destruction by this detergent. The incremental responses to activation by ions and substrates, and inhibition by ouabain, are consistent with the hypothesis that permeability-intact vesides of sarcolemma are present in the isolated preparation, and that detergent activation renders the vesides highly permeable to the ions, substrates, and ouabain.