乙二醇双（β-氨基乙醚）-N，N'-四乙酸钙络合物对肌浆网囊泡钙转运的刺激作用。

Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N',-tetraacetic acid.

作者信息

Berman M C

出版信息

J Biol Chem. 1982 Feb 25;257(4):1953-7.

Abstract

The calcium ion dependence of calcium transport by isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been investigated by means of the Calcium-stat method, in which transport may be measured in the micromolar free calcium ion concentration range, in the absence of calcium buffers. At pH 7.2 and 20 degrees C, ATP, in the range 1 to 10 mM, decreased [Ca2+]0.5 from 2.0 microM to 0.3 microM and decreased Vmax of oxalate-supported transport from 0.5 to 1.3 mumol min-1 mg-1. Simultaneous measurements of transport and of ATPase activity in the range 0.8 to 10 microM free Ca2+ showed a ratio of 2.1 calcium ions translocated/molecule of ATP hydrolyzed. Transport, in the presence of 5 mM ATP, ceased when calcium ion concentration fell to 0.6 to 1.2 microM, whilst ATPase activity of 90 nmol of ATP hydrolyzed min-1 mg-1 persisted. The data obtained by the Calcium-stat method differed from those described previously using calcium buffers, in that they showed lower apparent affinities of the transport site for calcium ions, more marked sigmoidal behavior, an effect of ATP concentration on Ca2+ concentration dependence and lower ATPase activity in the absence of transport. The calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) had no effect when transport was stimulated maximally at saturating free Ca2+ concentrations. However, at calcium ion levels below [Ca2+]0.5, 70 microM CaEGTA stimulated transport to rates of 20 to 45% of Vmax. Half-maximal stimulation of transport occurred at 19 microM CaEGTA. CaEGTA, 50 microM, decreased [Ca2+]0.5, determined at 5 mM ATP, from 1.3 microM to 0.45 microM. It is proposed that a ternary complex, E . Ca2+ . EGTA4-, is formed as an intermediate species during CaEGTA-stimulated calcium transport by sarcoplasmic reticulum membranes and stimulates the calcium pump at limiting free Ca2+ ion concentration.

摘要

采用钙稳态法研究了兔骨骼肌分离的肌浆网囊泡钙转运对钙离子的依赖性，该方法可在无钙缓冲液的情况下，在微摩尔游离钙离子浓度范围内测定转运。在pH 7.2和20℃条件下，1至10 mM范围内的ATP使[Ca2+]0.5从2.0 μM降至0.3 μM，并使草酸盐支持的转运的Vmax从0.5降至1.3 μmol min-1 mg-1。在0.8至10 μM游离Ca2+范围内同时测量转运和ATP酶活性，结果显示每水解1分子ATP转运2.1个钙离子。在5 mM ATP存在下，当钙离子浓度降至0.6至1.2 μM时转运停止，而90 nmol ATP水解min-1 mg-1的ATP酶活性仍持续存在。通过钙稳态法获得的数据与先前使用钙缓冲液描述的数据不同，在于它们显示转运位点对钙离子的表观亲和力较低、更明显的S形行为、ATP浓度对Ca2+浓度依赖性的影响以及在无转运时较低的ATP酶活性。当在饱和游离Ca2+浓度下最大程度刺激转运时，乙二醇双（β-氨基乙醚）-N,N,N',N'-四乙酸钙络合物（CaEGTA）没有作用。然而，在钙离子水平低于[Ca2+]0.5时，70 μM CaEGTA将转运刺激至Vmax的20%至45%。转运的半最大刺激发生在19 μM CaEGTA时。50 μM CaEGTA使在5 mM ATP下测定的[Ca2+]0.5从1.3 μM降至0.45 μM。有人提出，在CaEGTA刺激肌浆网膜钙转运过程中，三元复合物E·Ca2+·EGTA4-作为中间物种形成，并在有限的游离Ca2+离子浓度下刺激钙泵。