Tsuda T, Kaya S, Yokoyama T, Hayashi Y, Taniguchi K
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060, Japan.
J Biol Chem. 1998 Sep 18;273(38):24334-8. doi: 10.1074/jbc.273.38.24334.
Pig and dog kidney Na+,K+-ATPase preparations, irrespective of specific activity, showed approximately 0.5 mol of maximum phosphorylation/mol alpha-chain for ATP or acetyl phosphate (AcP) at steady state conditions. Pyridoxal 5'-diphospho-5'-adenosine (AP2PL)-treated pig kidney enzymes containing approximately 0.5 mol of AP2PL probe at Lys-480/mol (Tsuda, T., Kaya, S., Funatsu, H., Hayashi, Y., and Taniguchi, K. (1998) J. Biochem. (Tokyo) 123, 169-174) showed a quarter-site phosphorylation by ATP and half-site phosphorylation from AcP. The addition of 10 microM ATP to the Mg2+-Na+-bound AP2PL enzyme induced rapid quarter-site phosphorylation (47/s), followed by two different AP2PL fluorescence changes, a rapid decrease (29/s) and a slow increase (1.1/s). The addition of 1 mM AcP to the Mg2+-Na+-bound AP2PL enzyme induced a slow half-site phosphorylation (3/s), followed by a monophasic AP2PL fluorescence increase (1.2/s). After treatment of the AP2PL enzyme with fluorescein 5'-isothiocyanate to modify Lys-501 fully, the Mg2+-Na+-dependent phosphorylation capacity from ATP of the resulting AP2PL-fluorescein 5'-isothiocyanate enzyme was reduced to approximately 6% without significant changes in half-site phosphorylation capacity with respect to AcP, dynamic AP2PL fluorescence change by ATP and change by AcP. These data and others support the hypothesis that the functional membrane-bound Na+, K+-ATPase has tetrameric properties.
猪和狗肾的Na +，K + -ATP酶制剂，无论其比活性如何，在稳态条件下，对于ATP或乙酰磷酸（AcP），每摩尔α链显示约0.5摩尔的最大磷酸化。用吡哆醛5'-二磷酸-5'-腺苷（AP2PL）处理的猪肾酶，每摩尔赖氨酸-480含有约0.5摩尔的AP2PL探针（Tsuda，T.，Kaya，S.，Funatsu，H.，Hayashi，Y.和Taniguchi，K.（1998）J.Biochem.（东京）123,169 - 174）显示ATP引起的四分之一位点磷酸化和AcP引起的半位点磷酸化。向Mg2 + -Na +结合的AP2PL酶中添加10μMATP会诱导快速的四分之一位点磷酸化（47 / s），随后是两种不同的AP2PL荧光变化，快速下降（29 / s）和缓慢增加（1.1 / s）。向Mg2 + -Na +结合的AP2PL酶中添加1 mM AcP会诱导缓慢的半位点磷酸化（3 / s），随后是单相的AP2PL荧光增加（1.2 / s）。用5'-异硫氰酸荧光素处理AP2PL酶以完全修饰赖氨酸-501后，所得AP2PL-5'-异硫氰酸荧光素酶的Mg2 + -Na +依赖性ATP磷酸化能力降低至约6％，而相对于AcP的半位点磷酸化能力、ATP引起的动态AP2PL荧光变化和AcP引起的变化没有显著变化。这些数据和其他数据支持功能性膜结合Na +，K + -ATP酶具有四聚体性质的假设。
