Dissecting the Hydrolytic Activities of Sarcoplasmic Reticulum ATPase in the Presence of Acetyl Phosphate

F Soler, MI Fortea, A Lax, F Fernandez Belda
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Abstract

Sarcoplasmic reticulum vesicles and purified Ca$^{2+}$-ATPase hydrolyze acetyl phosphate both in the presence and absence of Ca$^{2+}$. The Ca$^{2+}$-independent activity was fully sensitive to vanadate, insensitive to thapsigargin, and proceeded without accumulation of phosphorylated enzyme. Acetyl phosphate hydrolysis in the absence of Ca$^{2+}$ was activated by dimethyl sulfoxide. The Ca$^{2+}$-dependent activity was partially sensitive to vanadate, fully sensitive to thapsigargin, and associated with steady phosphoenzyme accumulation. The Ca$^{2+}$/P(i) coupling ratio at neutral pH sustained by 10 mm acetyl phosphate was 0.57. Addition of 30% dimethyl sulfoxide completely blocked Ca$^{2+}$ transport and partially inhibited the hydrolysis rate. Uncoupling induced by dimethyl sulfoxide included the accumulation of vanadate-insensitive phosphorylated enzyme. When acetyl phosphate was the substrate, the hydrolytic pathway was dependent on experimental conditions that might or might not allow net Ca$^{2+}$ transport. The interdependence of both Ca$^{2+}$-dependent and Ca$^{2+}$-independent hydrolytic activities was demonstrated.
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剖析肉质网 ATP 酶在乙酰磷酸存在下的水解活动
肉质网囊泡和纯化的 Ca$^{2+}$-ATPase 在 Ca$^{2+}$ 存在和不存在的情况下都能水解磷酸乙酰。不依赖于 Ca$^{2+}$ 的活性对钒酸盐完全敏感,对硫代甘氨不敏感,并且没有磷酸化酶的积累。钙^{2+}$依赖性活性对瓦那酸盐部分敏感,对硫辛加精完全敏感,并与稳定的磷酸酶积累有关。在中性 pH 值下,由 10 毫米磷酸乙酰基维持的 Ca$^{2+}$/P(i) 耦合比为 0.57。加入 30% 的二甲基亚砜完全阻断了 Ca$^{2+}$ 的运输,并部分抑制了水解速率。二甲基亚砜诱导的解偶联包括对钒酸盐不敏感的磷酸化酶的积累。当以乙酰磷酸为底物时,水解途径取决于可能允许或不允许 Ca$^{2+}$ 净运输的实验条件。
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