心肌肌膜囊泡中依赖ATP的钠离子转运

ATP-dependent Na+ transport in cardiac sarcolemmal vesicles.

作者信息

Philipson K D, Nishimoto A Y

出版信息

Biochim Biophys Acta. 1983 Aug 24;733(1):133-41. doi: 10.1016/0005-2736(83)90099-8.

Abstract

Although the enzyme (Na+ + K+)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na+ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na+ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na+ transport sensitively. This technique relies on the presence of Na+-Ca2+ exchange and ATP-driven Na+ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na+ uptake is monitored by a subsequent Nai+-dependent Ca2+ uptake reaction (Na+-Ca2+ exchange) using 45Ca2+. We present evidence that the Na+-Ca2+ exchange will be linearly related to the prior active Na+ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na+ isotopes. Applying this method, we measure cardiac ATP-dependent Na+ transport and (Na+ + K+)-ATPase activities in identical ionic media. We find that the (Na+ + K+)-ATPase and the Na+ pump have identical dependencies on both Na+ and ATP. The dependence on [Na+] is sigmoidal, with a Hill coefficient of 2.8. Na+ pumping is half-maximal at [Na+] = 9 mM. The Km for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na+ pumping. This approach should allow other new investigations on ATP-dependent Na+ transport across cardiac sarcolemma.

摘要

尽管（Na⁺ + K⁺）-ATP酶已得到广泛表征，但在囊泡制剂中，关于其主要作用——ATP依赖性Na⁺泵转运的研究报道却很少。这是因为在大多数分离的膜系统中，准确测定同位素Na⁺的通量极其困难。利用高度纯化的心肌肌膜囊泡，我们开发了一种新技术，能够灵敏地检测ATP依赖性Na⁺转运的相对速率。该技术依赖于同一内翻式肌膜囊泡上存在的Na⁺-Ca²⁺交换和ATP驱动的Na⁺泵活性。通过随后利用⁴⁵Ca²⁺的Na⁺内依赖性Ca²⁺摄取反应（Na⁺-Ca²⁺交换）来监测ATP依赖性Na⁺摄取。我们提供的证据表明，Na⁺-Ca²⁺交换将与先前的活性Na⁺摄取呈线性相关。尽管此方法是间接的，但它比使用Na⁺同位素的直接方法灵敏得多。应用该方法，我们在相同的离子介质中测量心肌ATP依赖性Na⁺转运和（Na⁺ + K⁺）-ATP酶活性。我们发现，（Na⁺ + K⁺）-ATP酶和Na⁺泵对Na⁺和ATP具有相同的依赖性。对[Na⁺]的依赖性呈S形，希尔系数为2.8。在[Na⁺] = 9 mM时，Na⁺泵转运达到最大速率的一半。ATP的米氏常数为0.21 mM。ADP竞争性抑制ATP依赖性Na⁺泵转运。这种方法应能允许对跨心肌肌膜的ATP依赖性Na⁺转运进行其他新的研究。