轴突膜的（钠+，钾+）激活的三磷酸腺苷酶、协同性与调控。稳态分析
(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.
作者信息
Gache C, Rossi B, Lazdunski M
出版信息
Eur J Biochem. 1976 May 17;65(1):293-306. doi: 10.1111/j.1432-1033.1976.tb10417.x.
Abstract
- The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.
摘要
- ATP位点。已在非常广泛的Na⁺和K⁺浓度范围内研究了ATP位点之间的同促相互作用。在Na⁺（100 mM）和K⁺（10 mM）的标准浓度条件下，（Na⁺,K⁺）激活的ATP酶对ATP呈现米氏动力学。在极低的K⁺浓度下观察到负协同性时，稳态动力学行为会发生变化。通过研究在Na⁺和K⁺存在下ATP刺激的对硝基苯磷酸酯水解反应，清楚地证明了ATP存在高亲和力和低亲和力位点。在pH 7.5时，高亲和力和低亲和力位点对ATP的表观亲和力之比为86。2. Na⁺位点。Na⁺与其特定刺激位点（内部位点）的结合具有正协同性，希尔系数n(H(Na⁺)) = 2.0。Na⁺位点之间的同促相互作用不受K⁺浓度变化的影响。3. K⁺位点。(a) 通过在Na⁺和K⁺（有或无ATP）存在下跟踪（Na⁺,K⁺）-ATP酶活性以及对硝基苯磷酸酶活性，分析了K⁺与ATP酶的（外部）刺激位点的结合。结合的特征是希尔系数为1.0，K(0.5(K⁺)) = 0.1至0.8 mM。在5 mM至100 mM Na⁺之间不存在正协同性或负协同性。(b) 仅在K⁺存在下对对硝基苯磷酸酶或2,4-二硝基苯磷酸酶活性的分析表明存在具有正同促相互作用的K⁺低亲和力位点。在这种情况下的刺激特征为，K(0.5) = 5 mM，n(H) = 1.9。该位点家族的特性如下：首先，K⁺使低亲和力位点饱和会阻止ATP与其高亲和力内部位点结合。其次，K⁺低亲和力位点饱和会阻止Na⁺与其内部位点结合。第三，当Na⁺与其内部位点结合时，在ATP、对硝基苯磷酸酯或两种底物存在的情况下，这个位点家族会消失。Na⁺与其特定刺激位点的结合会促使形成K⁺的高亲和力类型位点。4. Mg²⁺对（Na⁺,K⁺）-ATP酶的刺激特征为希尔系数n(H(Mg²⁺)) = 1.0，K(0.5(Mg²⁺)) = 1 mM，刺激本质上是一种V效应。Mg²⁺与底物在各自位点的结合之间的异促效应很小。Mg²⁺、Na⁺和K⁺位点之间的异促相互作用也很小。5. 膜脂的流动性也控制着（Na⁺,K⁺）-ATP酶的活性。膜中的相变或分离几乎不影响底物、Na⁺、K⁺和Mg²⁺对膜两侧各自位点的识别特性。仅催化转化的速率受到影响。
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