Barrabin H, Fontes C F, Scofano H M, Nørby J G
Departamento de Bioquímica, ICB, CCS, Universidade Federal do Rio de Janeiro, Brasil.
Biochim Biophys Acta. 1990 Apr 13;1023(2):266-73. doi: 10.1016/0005-2736(90)90422-k.
Purified Na+, K(+)-ATPase was phosphorylated by [gamma-32P]ATP in a medium containing dimethylsulfoxide and 5 mM Mg2+ in the absence of Na+ and K+. Addition of K+ increased the phosphorylation levels from 0.4 nmol phosphoenzyme/mg of protein in the absence of K+ to 1.0 nmol phosphoenzyme/mg of protein in the presence of 0.5 mM K+. Higher velocities of enzyme phosphorylation were observed in the presence of 0.5 mM K+. Increasing K+ concentrations up to 100 mM lead to a progressive decrease in the phosphoenzyme (EP) levels. Control experiments, that were performed to determine the contribution to EP formation from the Pi inevitably present in the assays, showed that this contribution was of minor importance except at high (20-100 mM) KCl concentrations. The pattern of EP formation and its KCl dependence is thus characteristic for the phosphorylation of the enzyme by ATP. In the absence of Na+ and with 0.5 mM K+, optimal levels (1.0 nmol EP/mg of protein) were observed at 20-40% dimethylsulfoxide and pH 6.0 to 7.5. Addition of Na+ up to 5 mM has no effect on the phosphoenzyme level under these conditions. At 100 mM Na+ or higher the full capacity of enzyme phosphorylation (2.2 nmol EP/mg of protein) was reached. Phosphoenzyme formed from ATP in the absence of Na+ is an acylphosphate-type compound as shown by its hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the alpha-subunit of the Na+, K(+)-ATPase as demonstrated by acid polyacrylamide gel electrophoresis followed by autoradiography.
在不含Na⁺和K⁺的情况下，纯化的Na⁺,K⁺-ATP酶在含有二甲基亚砜和5 mM Mg²⁺的培养基中被[γ-³²P]ATP磷酸化。加入K⁺后，磷酸化水平从无K⁺时的0.4 nmol磷酶/mg蛋白质增加到存在0.5 mM K⁺时的1.0 nmol磷酶/mg蛋白质。在存在0.5 mM K⁺的情况下观察到酶磷酸化的速度更高。将K⁺浓度增加至100 mM会导致磷酶（EP）水平逐渐降低。为确定测定中不可避免存在的无机磷酸盐（Pi）对EP形成的贡献而进行的对照实验表明，除了在高（20 - 100 mM）KCl浓度下，这种贡献不太重要。因此，EP形成的模式及其对KCl的依赖性是ATP对该酶磷酸化的特征。在无Na⁺且有0.5 mM K⁺的情况下，在20 - 40%二甲基亚砜和pH 6.0至7.5时观察到最佳水平（1.0 nmol EP/mg蛋白质）。在这些条件下，加入高达5 mM的Na⁺对磷酶水平没有影响。在100 mM Na⁺或更高浓度时，达到了酶磷酸化的最大能力（2.2 nmol EP/mg蛋白质）。如对羟胺敏感性所示，在无Na⁺时由ATP形成的磷酶是一种酰基磷酸盐型化合物。酸性聚丙烯酰胺凝胶电泳后进行放射自显影表明，磷酸盐放射性被掺入到Na⁺,K⁺-ATP酶的α亚基中。
