Purification of cardiac sarcolemmal vesicles: high sodium pump content and ATP-dependent, calmodulin-activated calcium uptake.
Purification of cardiac sarcolemmal vesicles: high sodium pump content and ATP-dependent, calmodulin-activated calcium uptake.
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心脏肌膜囊泡的纯化：高钠泵含量和 ATP 依赖性、钙调蛋白激活的钙吸收。
DOI：
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发表时间：
1982
期刊：
影响因子：
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通讯作者：
T. Kanazawa
中科院分区：
文献类型：
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作者：
H. Kuwayama;T. Kanazawa
用密度梯度离心法从犬心室肌匀浆中制备了高纯度的心肌肌膜囊泡。该制剂显示出极高的（Na+，K+）-ATP酶含量。在Triton X-100和特定哇巴因结合的情况下，Triton X-100的存在下，Na+依赖性磷酸酶形成的稳态水平分别为773和907 pmol.mg-1的最佳条件下。另一方面，在不存在Triton X-100的情况下形成的Ca 2+依赖性磷酸酶的量小于2 pmol·mg-1。这表明该制剂实际上不含污染的肌浆网碎片。该制剂显示ATP依赖性的Ca 2+摄取。几乎所有的Ca 2+积累在添加ATP的迅速释放，随后加入NaCl。这一发现为心肌肌膜中存在ATP驱动的Ca ~（2+）泵提供了证据。2 μ M洋地黄毒苷、1 μ M莫能菌素和200 μ M二硝基苯酚不影响Ca 2+摄取。这些结果排除了Na+和H+的跨膜梯度参与这种Ca 2+吸收的可能性。Ca ~（2+）泵被钙调素激活。产生半最大激活的钙调蛋白浓度为0.05微克.ml-1，相当于3 nM。这种激活作用通过加入三氟拉嗪（一种钙调蛋白的特异性抑制剂）来消除。
Highly purified vesicles of cardiac sarcolemma were prepared from a homogenate of canine ventricular muscle by density gradient centrifugation. The preparation showed an extremely high content of (Na+,K+)-ATPase. The steady state levels of Na+-dependent phosphoenzyme formation in the presence of Triton X-100 and the specific ouabain binding in the absence of Triton X-100 were, respectively, 773 and 907 pmol.mg-1 under the optimum conditions. On the other hand, the amount of Ca2+-dependent phosphoenzyme formed in the absence of Triton X--100 was less than 2 pmol.mg-1. This demonstrates that the preparation was virtually free of contaminant sarcoplasmic reticulum fragments. The preparation showed ATP-dependent Ca2+ uptake. Almost all the Ca2+ accumulated on the addition of ATP was rapidly released by the subsequent addition of NaCl. This finding gives evidence that the ATP-driven Ca2+ pump exists in the cardiac sarcolemma. The Ca2+ uptake was unaffected by 2 microM digitoxin, 1 microM monesin, and 200 microM dinitrophenol. These results exclude the possibility that transmembrane gradients of Na+ and H+ were involved in this Ca2+ uptake. The Ca2+ pump was activated by calmodulin. The concentration of calmodulin giving a half-maximum activation was 0.05 micrograms.ml-1, which is equivalent to 3 nM. This activation was removed by addition of trifluoperazine, a specific inhibitor of calmodulin.