Tanaka N, Ogura T, Noguchi T, Hirano H, Yabe N, Hasunuma K
Kihara Institute for Biological Research, Yokohama City University, Japan.
J Photochem Photobiol B. 1998 Sep;45(2-3):113-21. doi: 10.1016/s1011-1344(98)00169-9.
To clarify the molecular mechanism for the transduction of light signals in plants, we have established an in vitro system that uses crude membrane and soluble fractions of stem sections of etiolated Pisum sativum L. cv. Alaska after irradiation by red light, or sequential application of red and far-red light to the stem section. In a previous report (T. Hamada et al., J. Photochem. Photobiol. B: Biol. 33 (1996) 143-151) the labelling of proteins in membrane fraction by [gamma-32P] ATP at 0 degree C for 15 s and subsequent separation of proteins by two-dimensional electrophoresis allowed unambiguous identification of a heavily phosphorylated protein spot at 18 kDa (p18). In the present study we have confirmed the former results in the membrane fraction, and obtained the result that an increase in the phosphorylation of p18 by red-light irradiation is observed in the soluble fraction. Further, we have provided evidence that the p18 in the soluble fraction is purified and identified as nucleoside diphosphate (NDP) kinase by Western blotting, immuno-precipitation, amino acid sequencing and cDNA analysis. Purified p18 shows autophosphorylation activity and strong phosphorylating activity against myelin basic protein (MBP), a substrate of MAP (mitogen activated protein) kinase. The results show that phytochrome-mediated light signals are transduced to NDP kinase, which may elicit signals by providing high concentrations of, for example, GTP from GDT and ATP, by the autophosphorylation and by the protein kinase activity similar to MAP kinase.
为阐明植物中光信号转导的分子机制，我们建立了一个体外系统，该系统使用经红光照射或对黄化豌豆（Pisum sativum L. cv. Alaska）茎段依次施加红光和远红光后的茎段粗膜和可溶部分。在之前的一份报告中（T. Hamada等人，《光化学与光生物学B：生物学》33 (1996) 143 - 151），在0℃下用[γ-32P]ATP对膜部分的蛋白质进行15秒标记，随后通过二维电泳分离蛋白质，从而明确鉴定出一个18 kDa的高度磷酸化蛋白质斑点（p18）。在本研究中，我们证实了膜部分的先前结果，并得到了在可溶部分中观察到红光照射使p18磷酸化增加的结果。此外，我们提供了证据，通过蛋白质免疫印迹、免疫沉淀、氨基酸测序和cDNA分析，可溶部分中的p18被纯化并鉴定为核苷二磷酸（NDP）激酶。纯化的p18显示出自身磷酸化活性以及对髓鞘碱性蛋白（MBP，一种丝裂原活化蛋白（MAP）激酶的底物）的强磷酸化活性。结果表明，光敏色素介导的光信号被转导至NDP激酶，该激酶可能通过自身磷酸化以及与MAP激酶相似的蛋白激酶活性，例如从GDP和ATP提供高浓度的GTP来引发信号。
