Alonso G, Walser M
J Gen Physiol. 1968 Jul;52(1):111-35. doi: 10.1085/jgp.52.1.111.
Rat brain microsomes, immobilized on a filter, were perfused with ATP-containing solutions in a device which made possible rapid change of perfusion media and frequent sampling of effluent. Inorganic phosphate production could be measured 10 times per sec. When ATP, sodium, or potassium was absent from the first perfusion medium and present in a second, and introduced without interrupting flow, phosphate output rose within a few tenths of a second. Inhibition by ouabain began within 0.3 sec but did not become maximal for at least 10 sec. Rapid binding of ouabain was minimal or absent, as was rapid release of ouabain on introducing potassium abruptly. Although the preparation bound some calcium reversibly, no measurable uptake of calcium occurred coincident with activation by ATP or by potassium, and no measurable release of calcium occurred coincident with the onset of ouabain inhibition. However, activation by sodium was consistently associated with simultaneous release (within < 1 sec) of calcium, averaging 46 pmole per mg of protein. Calcium release in response to sodium also occurred in the absence of ATP or in the presence of ouabain. At 0 degrees C sodium produced neither activation nor calcium release. The results are consistent with the possibility that sodium and calcium are competitively bound, even in the absence of ATP, to an active site on the enzyme distinct from the sites of potassium activation or glycoside inhibition.
固定在滤膜上的大鼠脑微粒体，在一种能够快速更换灌注介质并频繁采集流出液的装置中，用含ATP的溶液进行灌注。无机磷酸盐的生成每秒可测量10次。当第一种灌注介质中不存在ATP、钠或钾，而在第二种灌注介质中存在，并在不中断流动的情况下引入时，磷酸盐输出在十分之几秒内就会上升。哇巴因的抑制作用在0.3秒内开始，但至少10秒后才达到最大值。哇巴因的快速结合极少或不存在，突然引入钾时哇巴因的快速释放也是如此。尽管该制剂可逆地结合了一些钙，但在ATP或钾激活时未发生可测量的钙摄取，在哇巴因抑制开始时也未发生可测量的钙释放。然而，钠的激活始终与同时（在<1秒内）释放钙相关，平均每毫克蛋白质释放46皮摩尔钙。在没有ATP或存在哇巴因的情况下，对钠的反应也会发生钙释放。在0℃时，钠既不产生激活作用也不引起钙释放。这些结果与以下可能性一致：即使在没有ATP的情况下，钠和钙也竞争性地结合到酶上与钾激活位点或糖苷抑制位点不同的活性位点。
