配体对(Na~++K~+)-ATP酶E_2→E_1转变的影响

 本研究确定了在0℃条件下,(Na~++K~+)-ATP酶纯化制备物与5mmol/L Na~+或Mg~(2+)在5mmol/L咪唑(pH7.4)环境中预保温30分钟,然后进行磷酸化,可以获得最高磷酸化水平,Na~+或Mg~(2+)的K_(0.5)值分别为0.29mmol/L或0.35mmol/L;以ADP代替Na~+和Mg~(2+)与酶预保温,对E_2向E_1转变无任何影响,而与Na~+、Mg~(2+)一起存在时则能加强Na~+及Mg~2的预保温效果。

EFFECTS OF LIGANDS ON E_2 TO E_1 TRANSITION OF(Na~++ K~+)-ATPase

The purified (Na+ + K+) -ATPase from outer medulla of rabbit kidney is in an E2 conformation. In this study the optimal preincubation conditions for promoting E2 to E1 transition were investigated. On addition of Na+ or Mg2+ in various concentrations the enzyme was preincubated for 30 min at 0℃ in 5mmol/L imidazole (pH 7.4). After preincubation the enzyme was subjected to phosphorylation under the optimal conditions. 5mmol/L Na+ or Mg2+ and 5mmol/L imidazole (pH 7.4) were the optimal preincubation conditions necessary to accomplish a complete shift of the equilibrium to the E1 conformation, the half-maximal concentrations were 0.29mmol/L Na+ and 0.35mmol/L Mg2+. Preincubation of enzyme for 30 min with up to 50μmol/L ADP did not induce any measurable transition from E2 to E1, the rate of transition of E2 to E, was greater when ADP was present together with Na+ and Mg2+ than when ADP was absent. The results indicated that substantial fraction of the native enzyme in the preparation usually existed in the form of E2 conformation, and was shift to the E1 conformation after preincubation.