Schuurmans Stekhoven F M, Swarts H G, de Pont J J, Bonting S L
Biochim Biophys Acta. 1985 Apr 26;815(1):16-24. doi: 10.1016/0005-2736(85)90468-7.
A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.
在50 mM咪唑 - HCl（pH 7.0）中，在未添加Na⁺和K⁺且存在10 - 100 μM Mg²⁺的情况下，观察到较高的基础磷酸化水平（约为最佳Na⁺依赖性磷酸化水平的70%）。在50 mM Tris - HCl（pH 7.0）中，无论Mg²⁺浓度如何，基础水平仅为5%。然而，咪唑作为磷酸化的激活剂不如Na⁺有效（在可比条件下，咪唑 - H⁺的Km为5.9 mM，Na⁺的Km为2 mM）。咪唑激活的磷酸化强烈依赖于pH，在pH小于或等于7时最佳，在pH大于或等于8时最低，而Na⁺激活的磷酸化在pH 7.4时最佳。这表明咪唑 - H⁺是激活物种。咪唑促进Na⁺刺激的磷酸化。Na⁺的Km从5 mM咪唑 - HCl时的0.63 mM降至50 mM咪唑 - HCl（pH 7；所有情况下为0.1 mM Mg²⁺）时的0.21 mM。咪唑激活的磷酸化比Na⁺激活的磷酸化对K⁺抑制更敏感（I50 = 12.5 μM对I50 = 180 μM）。Mg²⁺拮抗咪唑 - H⁺的激活以及K⁺的抑制。Mg²⁺的Ki值（约0.3 mM）对于这两种拮抗作用是相同的。Tris缓冲液（pH 7.0）在50 mM咪唑 - HCl（pH 7.0）加0.1 mM Mg²⁺中以30 mM的I50值抑制咪唑激活的磷酸化。我们得出结论，咪唑 - H⁺而非Tris - H⁺可以替代Na⁺作为ATP依赖性磷酸化的激活剂，主要是通过将E2----E1转变向右移动，导致一种与Tris缓冲液中不同的磷酸化E1构象。
