Two-step internalization of calcium from a single E.apprx.P.cntdot.Ca2 species by the calcium ATPase
Two-step internalization of calcium from a single E.apprx.P.cntdot.Ca2 species by the calcium ATPase
复制标题
钙 ATP 酶对单个 E.apprx.P.cntdot.Ca2 物种中钙的两步内化
DOI：
--
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
W. Jencks
中科院分区：
文献类型：
--
作者：
D. Khananshvili;W. Jencks
肌浆网小泡(SRV)在5-10ms内被ATP磷酸化，导致2*Ca~(2+)/EPtot[单独被乙二醇双(β-氨基乙基醚)-N，N，N‘，N’-四乙酸(EGTA)猝灭]在“空”(10微米游离钙内)或“负载”(20-40 mm内游离钙内)中均被阻断。通过使用ADP+EGTA猝灭来测量从闭塞的E大约P。*Ca~(2+)“内化”的速率；未被该猝灭去除的*Ca~(2+)离子被定义为内化。在SRV内存在20-40 mM未标记的Ca~(2+)时，1~*Ca~(2+)/EPtot由~(45)Ca~(2+)标记的E内化，约为P~(2+)，其一级速率常数KL=34 S~(-1)。在相同的初始速率下，空SRV占2*Ca~(2+)/EPtot，但总的速率常数为kobsd=17 S~(-1)。Ca~(2+)内化的表观速率常数(kb=17 S~(-1))被[Ca]In抑制，K_(0.5)约1.3 mm，希尔系数n=1.1。这些数据表明，这两个钙离子是按顺序内化的，推测是从通道中不同的顺序位置内化的。[32P]由E.Ca2与[伽马-32P]三磷酸腺苷和EGTA快速混合获得的Ep·Ca~(2+)以两相时间过程消失，滞后约为34 S-1，随后Ep*衰变，速率常数约为17 S-1。这表明，在酶改变其催化磷酸转移到水而不是ADP的专一性之前，这两个钙离子必须内化。当ATP浓度从0.25mM增加到3 mM时，45Ca~(2+)内化速率从34增加到69·S~(-1)，第二Ca~(2+)从17增加到34 S~(-1)。高[ATP]也加速了[~(32)P]Ep的两个时相，钙消失的速度相同。这些数据与一种单一形式的ADP敏感E一致，大约是P.Ca2，它顺序地内化两个离子。囊泡内的体积估计为2.0微L/mg，因此酶的一次周转产生4 mM的内部[Ca~(2+)]。
Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone] in both "empty" (10 microM free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E approximately P.*Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot is internalized from 45Ca-labeled E approximately P.*Ca2 with a first-order rate constant of kl = 34 s-1. Empty SRV take up 2 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 approximately 1.3 mM and a Hill coefficient of n = 1.1. These data show that the two Ca2+ ions are internalized sequentially, presumably from separate sequential sites in the channel. [32P]EP.Ca2 obtained by rapid mixing of E.Ca2 with [gamma-32P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to approximately 34 s-1, followed by EP* decay with a rate constant of approximately 17 s-1. This shows that both Ca2+ ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of 45Ca2+ internalization from 34 to 69 s-1 for the first Ca2+ and from 17 to 34 s-1 for the second Ca2+. High [ATP] also accelerates both phases of [32P]EP.Ca2 disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E approximately P.Ca2 that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 microL/mg, so that one turnover of the enzyme gives 4 mM internal [Ca2+].
登录
查看更多内容
调节核苷酸位点与肌浆网 Ca2-ATP 酶催化位点的关系。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Bishop,JE;Al-Shawi,MK;Inesi,G
通讯作者：
Inesi,G
ATP 从钙 ATP 酶中缓慢解离。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Pickart,CM;Jencks,WP
通讯作者：
Jencks,WP
兔骨骼肌浆网钙依赖性三磷酸腺苷反应中磷酸酶转化的瞬态动力学。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Wang,T
通讯作者：
Wang,T
DOI：
--
发表时间：
1981
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ikemoto,N;Garcia,AM;Kurobe,Y;Scott,TL
通讯作者：
Scott,TL
E1-E2 模型未能令人满意地描述肌浆网钙三磷酸腺苷酶与 5-三磷酸腺苷和 Ca2 的反应。
影响因子：
2.9
作者：
Stahl,N;Jencks,WP
通讯作者：
Jencks,WP