Hamada T, Tanaka N, Noguchi T, Kimura N, Hasunuma K
Yokohama City University, Kihara Institute for Biological Research, Japan.
J Photochem Photobiol B. 1996 Apr;33(2):143-51. doi: 10.1016/1011-1344(95)07236-5.
The molecular mechanism of light signal perception via phytochrome was analysed using the third internodes of etiolated pea seedlings irradiated with red or red followed by far-red light. A crude membrane fraction prepared from the tissue was labelled by [gamma-32P]ATP at 4 x 10(-8) M for 15 s at 0 degree C, and the proteins were separated by two-dimensional gel electrophoresis. The phosphorylation of a protein with a molecular mass of about 15 kDa in the crude membrane fraction increased with an increase in the intensity of red light irradiation (10, 50 and 100 mumol m-2 s-1) for 20 s. Successive irradiation with red light (100 mumol m-2 s-1 for 20 s) and subsequent irradiation with far-red light reduced the phosphorylation of the protein, depending on the intensity of the far-red light (from 0.1 to 8 mumol m-2 s-1 for 180 s). A plasma membrane purified from the crude membrane fraction from red light irradiated tissue showed a rapid phosphorylation of the 15 kDa protein by 4 x 10(-8) M [gamma-32P]ATP at 0 degree C for 7 s, and subsequent addition of ATP, GTP, ADP or GDP at 10(-5) or 10(-6) M efficiently removed the phosphoryl group of the 15 kDa protein. The 15 kDa protein was autophosphorylated in the gel following separation by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The partially purified 15 kDa protein included nucleoside diphosphate kinase (NDP kinase) activity, as well as cross-reactivity with an antibody specific to rat NDP kinase as assayed by immunostaining and crossreactivity with an antibody specific to ricet NDP kinase as assayed by immunoprecipitation.
利用黄化豌豆幼苗的第三节间，用红光或红光继之以远红光照射，分析了通过光敏色素感知光信号的分子机制。从该组织制备的粗膜部分在0℃下用4×10⁻⁸M的[γ-³²P]ATP标记15秒，然后通过二维凝胶电泳分离蛋白质。粗膜部分中一种分子量约为15kDa的蛋白质的磷酸化随着红光照射强度（10、50和100μmol m⁻² s⁻¹）增加20秒而增加。先用红光（100μmol m⁻² s⁻¹照射20秒）连续照射，随后用远红光照射，会使该蛋白质的磷酸化减少，这取决于远红光的强度（0.1至8μmol m⁻² s⁻¹照射180秒）。从红光照射组织的粗膜部分纯化的质膜在0℃下用4×10⁻⁸M的[γ-³²P]ATP快速磷酸化15kDa蛋白质7秒，随后加入10⁻⁵或10⁻⁶M的ATP、GTP、ADP或GDP能有效去除15kDa蛋白质的磷酸基团。15kDa蛋白质在十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳分离后在凝胶中发生自磷酸化。部分纯化的15kDa蛋白质具有核苷二磷酸激酶（NDP激酶）活性，通过免疫染色检测与大鼠NDP激酶特异性抗体有交叉反应，通过免疫沉淀检测与水稻NDP激酶特异性抗体有交叉反应。
