Regulation of steady state filling in sarcoplasmic reticulum. Roles of back-inhibition, leakage, and slippage of the calcium pump.
Regulation of steady state filling in sarcoplasmic reticulum. Roles of back-inhibition, leakage, and slippage of the calcium pump.
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DOI：
10.1016/s0021-9258(18)83639-0
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发表时间：
1989-04
期刊：
影响因子：
--
通讯作者：
G. Inesi;L. Meis
中科院分区：
文献类型：
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作者：
G. Inesi;L. Meis
在稳定状态下，肌浆网囊泡的钙填充由于草酸盐的沉淀而大大增加。我们发现低浓度（1mm）的pio通过与管腔钙形成可溶性复合物也允许更大的负荷，这种效应可能与生理相关。此外，ATP再生系统清除ADP通过防止泵的逆转有利于钙负荷。我们还发现atp酶和转运活性的解偶联是限制钙负荷的另一个因素。事实上，钙的吸收和ATP的利用在加入ATP后的瞬时状态下以2:1的摩尔比发生，但在稳定状态下减少到更低的值。即使没有高导电性通道（仅存在于“重”囊泡中），“轻”囊泡也会出现钙泄漏，这种钙泄漏在atp酶激活的浓度范围内被介质Ca2+抑制，并且可能与参与钙运输的atp酶通道有关。很明显，在atp酶周转的条件下，在高腔内Ca2+和ADP的存在下，钙通过该通道的滑移产生了催化和运输活性的真正解耦。通过腔内Ca2+络合和ATP再生改善偶联，并受反应介质溶剂特性的影响。通过多步反应机制的稳态分析，阐明了腔内Ca2+和ADP的协同作用，以及磷酸酶裂解的替代途径的作用。结论是，理想的（2:1）转运和atp酶活性的化学计量偶联并不是由一个强制性的催化途径保证的（正如迄今为止发表的所有反应方案所预测的那样）；相反，偶联受配体浓度及其对二级反应的影响以及由此产生的中间态分布的影响。
Calcium filling of sarcoplasmic reticulum vesicles in the steady state is greatly increased by precipitation of lumenal calcium with oxalate. We find that low concentrations (1 mM) of Pialso allow greater loading by forming a soluble complex with lumenal calcium, an effect that is likely to be of physiological relevance. Furthermore, ADP scavenging by ATP regenerating systems favors calcium loading by preventing reversal of the pump. We also find that uncoupling of ATPase and transport activities is another factor limiting calcium loading. In fact, calcium uptake and ATP utilization occur with a molar ratio of 2:1 in the transient state following addition of ATP but decrease to much lower values in the steady state. Even in the absence of the highly conductive channel which is present only in “heavy” vesicles, “light” vesicles display calcium leakage which is inhibited by medium Ca2+ in the concentration range of ATPase activation and is likely related to an ATPase channel which is involved in calcium transport. It is apparent that, under conditions of ATPase turnover and in the presence of high lumenal Ca2+and ADP, slippage of calcium through this channel produces true uncoupling of catalytic and transport activities. Coupling is improved by complexation of lumenal Ca2+and by ATP regeneration and is influenced by the solvent characteristics of the reaction medium. The synergistic effects of lumenal Ca2+and ADP, and the role of alternate pathways for phosphoenzyme cleavage, are clarified by steady state analysis of a multiple step reaction mechanism. It is concluded that the ideal (2:1) stoichiometric coupling of transport and ATPase activities is not insured by an obligatory pathway of catalysis (as predicted by all reaction schemes published so far); rather, coupling is influenced by the concentrations of ligands and their effects on second order reactions and the consequent distribution of intermediate states.