Kaya S, Tsuda T, Hagiwara K, Fukui T, Taniguchi K
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
J Biol Chem. 1994 Mar 11;269(10):7419-22.
The Lys-480 in the alpha-subunits of Na+,K(+)-ATPase from pig kidneys was specifically modified with pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probes in the presence of NaCl. The site was shown to be the same as the ATP-protectable binding of these probes (Hinz, H.R., and Kirley, T.L. (1990) J. Biol. Chem. 265, 10260-10265). Modifications strongly reduced both Na+,K(+)-ATPase activity and the amount of Na(+)-dependent phosphoenzyme from [32P]ATP but not from [32P]acetyl-phosphate (AcP). Addition of AcP to the enzyme induced a slight decrease in the fluorescence of the PLP probe in the presence of 2 M NaCl and 4 mM MgCl2 but a single exponential increase in the presence of 16 mM NaCl and 4 mM MgCl2. The addition of ATP induced single exponential fluorescence increases at both Na+ concentrations. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the formation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP2PL probes at Lys-480 in the presence of Na+ and Mg2+ do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes but that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg2+.
在氯化钠存在的情况下，用磷酸吡哆醛（PLP）或磷酸吡哆醛 - 5'-二磷酸 - 5'-腺苷（AP2PL）探针特异性修饰猪肾Na⁺,K⁺-ATP酶α亚基中的赖氨酸480位点。结果表明该位点与这些探针的ATP可保护结合位点相同（欣茨，H.R.，和柯利，T.L.（1990年）《生物化学杂志》265卷，10260 - 10265页）。修饰作用显著降低了Na⁺,K⁺-ATP酶的活性以及由[³²P]ATP而非[³²P]乙酰磷酸（AcP）形成的Na⁺依赖性磷酸酶的量。在2 M氯化钠和4 mM氯化镁存在的情况下，向酶中添加AcP会导致PLP探针的荧光略有下降，但在16 mM氯化钠和4 mM氯化镁存在的情况下会导致荧光呈单指数增加。在两种钠离子浓度下，添加ATP都会导致荧光呈单指数增加。数据表明，这些探针能够感知与AcP诱导的磷酸酶形成相关的分子事件，大概也能感知与Mg-Na-ATP-酶复合物形成相关的分子事件。数据还表明，在Na⁺和Mg²⁺存在的情况下，赖氨酸480位点的PLP或AP2PL探针不会影响从AcP到天冬氨酸369的转磷酸化以形成磷酸酶，但它们会抑制来自ATPγ-磷酸基团的转磷酸化以及在没有Mg²⁺时的ATP结合。
