STUDIES ON THE DARK/LIGHT REGULATION OF MAIZE LEAF PYRUVATE, ORTHO-PHOSPHATE DIKINASE BY REVERSIBLE PHOSPHORYLATION
STUDIES ON THE DARK/LIGHT REGULATION OF MAIZE LEAF PYRUVATE, ORTHO-PHOSPHATE DIKINASE BY REVERSIBLE PHOSPHORYLATION
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DOI：
10.1016/0003-9861(85)90503-x
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发表时间：
1985-01-01
影响因子：
3.9
通讯作者：
CHOLLET, R
中科院分区：
文献类型：
--
作者：
BUDDE, RJA;HOLBROOK, GP;CHOLLET, R
在玉米叶片中，丙酮酸正磷酸二激酶（PPDK）在黑暗中失活，在光照下重新激活。使用纯化的PPDK和部分纯化的玉米调节蛋白的体外研究证实了先前的报道，该报道将失活/再活化与苏氨酰残基的可逆磷酸化/去磷酸化相关联。通过监测稳定性的外源性32 P标记的腺苷酸底物在失活过程中，我们已经牢固地建立了ADP作为特定的磷酸供体。在离体的玉米叶肉原生质体预照射32 Pi，我们观察到一个三至五倍高的PPDK活性原位在光中，和相应的三至五倍高水平的磷酸化的94 kDa的PPDK原聚体在黑暗中。基于HPLC的磷酸氨基酸分析的PPDK纯化的玉米叶片的光和暗适应植物揭示了P-丝氨酸的存在。来自暗适应植物的失活酶（体内失活）也含有P-苏氨酸。从光适应植物的叶片中纯化的PPDK的总磷酸盐含量约为0.5mol/mol原体，从暗适应植物的叶片中纯化的PPDK的总磷酸盐含量约为1.5mol/mol原体。由于从光适应（活性PPDK）和暗适应（失活PPDK）植物纯化的酶之间的差异在于后者中存在P-苏氨酸，这表明1 mol P-苏氨酸/mol 94-kDa启动子的体内失活化学计量。这些互补的研究与玉米叶PPDK在体外，原位，和在体内提供了令人信服的证据，暗/光调节这一关键的C4-光合作用酶的可逆磷酸化。
In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa promoter. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation.