Martín-Romero F J, García-Martín E, Gutiérrez-Merino C
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Biochim Biophys Acta. 1996 Aug 14;1283(1):51-9. doi: 10.1016/0005-2736(96)00074-0.
The ecto-ATPase activity of synaptosomes plasma membrane decays exponentially as a function of time from 0.35 +/- 0.05 to 0.08 +/- 0.02 mumol ATP hydrolyzed per min per mg synaptosome protein. The first-order rate constant of inactivation is dependent on the Mg-ATP concentration varying from 0.042 +/- 0.001 min-1 with 30 microM ATP up to 0.216 +/- 0.003 min-1 with 2 mM ATP. The non-hydrolyzable ATP analogue, beta-gamma-methyleneadenosine 5'-triphosphate, did not produce inactivation of the ecto-ATPase activity. Thus, the inactivation of the ecto-ATPase activity requires hydrolysis of ATP. Product inhibition can be excluded because ADP, AMP, adenosine and inorganic phosphate up to 1 mM had no effect on the inactivation of the ecto-ATPase. Concanavalin A partially protected against the ATP-dependent inactivation. The ecto-ATPase inactivation produced by Mg-ATP is partially reverted by centrifugation, removal of the supernatant and resuspension of synaptosomes in a fresh medium. This partial reversion occurs in parallel to the release to the supernatant of phophorylated protein(s) of 90-95 kDa. Alkaline phosphatase treatment fully reverts the ecto-ATPase inactivation. We conclude that the ATP-induced inactivation is mediated, at least partially, by phosphorylation of membrane proteins.
突触体质膜的胞外ATP酶活性随时间呈指数衰减，从每分钟每毫克突触体蛋白水解0.35±0.05微摩尔ATP降至0.08±0.02微摩尔ATP。失活的一级速率常数取决于Mg-ATP浓度，范围从30微摩尔ATP时的0.042±0.001分钟⁻¹到2毫摩尔ATP时的0.216±0.003分钟⁻¹。不可水解的ATP类似物β-γ-亚甲基腺苷5'-三磷酸不会导致胞外ATP酶活性失活。因此，胞外ATP酶活性的失活需要ATP水解。由于高达1毫摩尔的ADP、AMP、腺苷和无机磷酸盐对胞外ATP酶的失活没有影响，所以可以排除产物抑制作用。伴刀豆球蛋白A对ATP依赖性失活有部分保护作用。Mg-ATP导致的胞外ATP酶失活通过离心、去除上清液并将突触体重悬于新鲜培养基中可部分恢复。这种部分恢复与90 - 95 kDa磷酸化蛋白释放到上清液中同时发生。碱性磷酸酶处理可完全恢复胞外ATP酶的失活。我们得出结论，ATP诱导的失活至少部分是由膜蛋白磷酸化介导的。
