Blostein R
J Biol Chem. 1975 Aug 10;250(15):6118-24.
When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.
当人红细胞膜在0℃下用极低浓度的[γ-32P]ATP（0.02μM）进行磷酸化，然后加入EDTA时，可观察到Na +ATP酶的磷酸化酶中间体迅速消失。磷酸化酶消失的初始快速阶段在很大程度上与P1释放无关，其速率常数kD比稳态时Na +ATP酶活性与磷酸化酶中间体的比率v:EP大几倍。得出的结论是，这种磷酸化酶的快速消失是由于通过磷酸化逆转重新合成ATP所致。相反，当加入过量的非放射性ATP以减少E32P形成时，如果存在Mg2 +，则未观察到快速逆转；然而，与ATP一起加入的K +会刺激逆转。当使用较高浓度的ATP（大于或等于10(-6)M）对酶进行磷酸化时，加入EDTA后也不太可能出现快速逆转，因为在较高的ATP浓度下，kD与v:EP相当。这些结果与Na +ATP酶由两个或更多催化亚基组成的概念相符，其中一个催化位点的ATP可以调节另一个位点的反应活性。
