Starling A P, Dalton K A, East J M, Oliver S, Lee A G
Department of Biochemistry, University of Southampton, Hants, U.K.
Biochem J. 1996 Nov 15;320 ( Pt 1)(Pt 1):309-14. doi: 10.1042/bj3200309.
ATPase activities for the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted into dioleoylphosphatidylethanolamine [di(C18:1)PE] are, at temperatures higher than 20 degrees C, lower than in dioleoylphosphatidylcholine [di(C18:1)PC], whereas in egg yolk phosphatidylethanolamine the activities are the same as in di(C18:1)PC up to 25 degrees C, suggesting that low ATPase activities occur when the phosphatidylethanol-amine species is in the hexagonal H11 phase. ATPase activities measured in mixtures of di(C18:1)PC and di(C18:1)PE do not change with changing di(C18:1)PE content up to 80%. It is concluded that curvature frustration in bilayers containing di(C18:1)PE has no effect on ATPase activity. The rates of phosphorylation and of Ca2+ transport are identical for the native ATPase and for the ATPase in di(C18:1)PE. Dephosphorylation of the phosphorylated ATPase in di(C18:1)PE at 25 degrees C is, however, slower than for the native ATPase, explaining the lower steady-state rate of ATP hydrolysis; in egg yolk phosphatidylethanolamine at 25 degrees C the rate of dephosphorylation is equal to that for the unreconstituted ATPase. Phosphorylation of the ATPase by P1 in the absence of Ca2+ is unaffected by reconstitution in di(C18:1)RE. The stoichiometry of Ca2+ binding to the ATPase is also unaltered. Studies of the effect of di(C18:1)PE on the fluorescence intensity of the ATPase labelled with 7-chloro-4-nitro-2,1,3-benzoxadiazole are consistent with an increase in the E1/E2 equilibrium constant, where E1 is the conformation of the ATPase with two high-affinity binding sites for Ca2+ exposed to the cytoplasm, and E2 is a conformation unable to bind cytoplasmic Ca2+. A slight increase in affinity for Ca2+ can be attributed to the observed increase in the E1/E2 equilibrium constant.
重构于二油酰磷脂酰乙醇胺[二（C18:1）PE]中的骨骼肌肌浆网Ca(2+)-ATP酶，在高于20℃的温度下，其ATP酶活性低于重构于二油酰磷脂酰胆碱[二（C18:1）PC]中的情况，而在蛋黄磷脂酰乙醇胺中，直至25℃其活性与二（C18:1）PC中的相同，这表明当磷脂酰乙醇胺处于六方H11相时会出现低ATP酶活性。在二（C18:1）PC和二（C18:1）PE的混合物中测得的ATP酶活性，在二（C18:1）PE含量高达80%时不会随其含量变化而改变。得出的结论是，含有二（C18:1）PE的双层膜中的曲率受挫对ATP酶活性没有影响。天然ATP酶和二（C18:1）PE中的ATP酶的磷酸化速率和Ca2+转运速率相同。然而，二（C18:1）PE中磷酸化的ATP酶在25℃时的去磷酸化比天然ATP酶慢，这解释了ATP水解的稳态速率较低的原因；在25℃的蛋黄磷脂酰乙醇胺中，去磷酸化速率与未重构的ATP酶相同。在没有Ca2+的情况下，P1对ATP酶的磷酸化不受重构于二（C18:1）RE中的影响。Ca2+与ATP酶结合的化学计量也未改变。对二（C18:1）PE对用7-氯-4-硝基-2,1,3-苯并恶二唑标记的ATP酶荧光强度的影响的研究，与E1/E2平衡常数的增加一致，其中E1是ATP酶的一种构象，有两个高亲和力的Ca2+结合位点暴露于细胞质中，而E2是一种无法结合细胞质Ca2+的构象。对Ca2+亲和力的轻微增加可归因于观察到的E1/E2平衡常数的增加。
