肌浆网Ca2+-ATP酶功能活性中两个高亲和力钙位点的作用差异

Distinction of the roles of the two high-affinity calcium sites in the functional activities of the Ca2+-ATPase of sarcoplasmic reticulum.

作者信息

Scott T L, Shamoo A E

出版信息

Eur J Biochem. 1984 Sep 3;143(2):427-36. doi: 10.1111/j.1432-1033.1984.tb08390.x.

DOI:10.1111/j.1432-1033.1984.tb08390.x

Abstract

The effects of trypsin digestion and low temperature on Ca2+ binding and on Ca2+ activation of ATP hydrolysis by the high-affinity transport sites of the Ca2+, Mg2+-ATPase of sarcoplasmic reticulum were examined. Sarcoplasmic reticulum vesicles contain 0.7-1.1 high-affinity Ca2+ sites per 10(5) g sarcoplasmic reticulum with K = 3-5 X 10(5) M-1, as well as sites of lower affinity. The first cleavage of the ATPase with trypsin (TD1) has no effect on the binding properties of the high affinity sites. The second tryptic cleavage (TD2) decreases the affinity of the high sites to K = 3 X 10(4) M-1 with conservation of the total number of sites. The purified ATPase contains 1.6-2.0 high affinity Ca2+ sites per 10(5) g protein when measured at 23 degrees C, while at 0-4 degrees C there is approximately equal to 1 high-affinity (K = 5 - 10 X 10(5) M-1) affinity site and approximately equal to 1 intermediate-affinity (K = 3 X 10(4) M-1) site per 10(5) g. Trypsin digestion to the point of TD1 has no effect on either the number or the binding constants of the high-affinity sites. Upon TD2 cleavage, one of the sites is converted to the intermediate-affinity state, while the other remains at high affinity. After TD2 modification of the enzyme both of the sites are in the intermediate affinity state at 4 degrees C. On the basis of the binding data, several models for the roles of the Ca2+ sites in the activation of ATP hydrolysis are derived. The results are summarized by a scheme in which the two high-affinity Ca2+ sites are heterogeneous with respect to sensitivity to temperature and to TD2 modification. The results of this and a previous study [Scott, T. L. and Shamoo, A. E. (1982) J. Membr. Biol. 64, 137-144] indicate that while occupation of either of the two Ca2+ sites can stimulate ATP hydrolysis, the site which is sensitive to TD2 is essential for the coupling of hydrolysis to Ca2+ transport.

摘要

研究了胰蛋白酶消化和低温对肌浆网Ca²⁺、Mg²⁺ - ATP酶高亲和力转运位点的Ca²⁺结合及Ca²⁺激活ATP水解的影响。肌浆网囊泡每10⁵g肌浆网含有0.7 - 1.1个高亲和力Ca²⁺位点，K = 3 - 5×10⁻⁵M⁻¹，还有低亲和力位点。用胰蛋白酶对ATP酶进行第一次切割（TD1）对高亲和力位点的结合特性没有影响。第二次胰蛋白酶切割（TD2）使高亲和力位点的亲和力降至K = 3×10⁻⁴M⁻¹，而位点总数不变。纯化的ATP酶在23℃下测量时，每10⁵g蛋白质含有1.6 - 2.0个高亲和力Ca²⁺位点，而在0 - 4℃时，每10⁵g约有1个高亲和力（K = 5 - 10×10⁻⁵M⁻¹）位点和约1个中等亲和力（K = 3×10⁻⁴M⁻¹）位点。胰蛋白酶消化至TD1程度对高亲和力位点的数量或结合常数均无影响。在TD2切割后，其中一个位点转变为中等亲和力状态，而另一个保持高亲和力。在酶经TD2修饰后，在4℃时两个位点均处于中等亲和力状态。根据结合数据，推导了几种Ca²⁺位点在激活ATP水解中作用的模型。结果用一个示意图总结，其中两个高亲和力Ca²⁺位点在对温度和TD2修饰的敏感性方面是异质的。本研究及之前一项研究[斯科特，T. L. 和沙穆，A. E.（1982年）《膜生物学杂志》64卷，137 - 144页]的结果表明，虽然占据两个Ca²⁺位点中的任何一个都能刺激ATP水解，但对TD2敏感的位点对于水解与Ca²⁺转运的偶联至关重要。