A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification.
A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification.
复制标题
燕麦光敏色素中的光可逆圆二色性光谱变化被结合在其 N 末端附近的单克隆抗体和发色团修饰所抑制。
DOI：
10.1021/bi00390a010
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Pratt,LH
中科院分区：
文献类型：
--
作者：
Chai,YG;Song,PS;Cordonnier,MM;Pratt,LH
材料与方法光敏色素的纯化。如前所述纯化未降解的124-kDa光敏色素（Chai和Song，1986）。简单地说，通过硫酸铵沉淀和羟基磷灰石（HA）层析从4天龄黄化燕麦幼苗的提取物中部分纯化光敏色素。然后对HA纯化的光敏色素进行硫酸铵反萃取。将上清液部分（II）加到Bio-Gel A-1.5 μ m柱上，该柱预先用含有1 mMEDTA的20 mMKPB，pH7.8（4 ℃）平衡。用相同的缓冲液从柱上洗脱光敏色素。用该方法常规获得SAR值为0.98-1.13的光敏色素（Chai等人，1987年）。本文报道的所有实验均用光敏色素进行，除非另有说明，光敏色素在4 ℃下在绿色安全光下进行，光敏色素通过用Bausch和Lomb显微镜照明器结合666 nm干涉滤光片（Oriel C572-6600）进行红光照射和用远红截止滤光片（Ealing 26 -4457）进行远红照射进行光转换。通量分别为7.5W/m2和1.6kW/m2。
Materials and MethodsPhytochrome Purification. Undegraded, 124-kDa phytochrome was purified as previously described (Chai & Song, 1986). Briefly, phytochrome was partially purified from ex-tracts of 4-day-old etiolated oat seedlings by ammonium sulfate precipitation and hydroxyapatite (HA) chromatography. The HA-purified phytochrome was then subjected to ammonium sulfate back-extraction. A supernatant fraction (II) was ap-plied to a Bio-Gel A-1.5 m column, which was previously equilibrated with 20 mM KPB, pH 7.8 (4 C), containing 1 mM EDTA. Phytochrome was eluted from the column with the same buffer. Phytochrome with SAR values of 0.98-1.13 was routinely obtained with this procedure (Chai et al., 1987). All experiments reported here were performed with phyto-chrome of SAR= 1.13 and under green safety light at 4 C, unless otherwise noted.Phytochrome was photoconverted by irradiating with a Bausch and Lomb microscope illuminator combined with a 666-nm interference filter (Oriel C572-6600) for red light, and with a far-red cutoff filter (Ealing26-4457) for far-red irra-diation. Fluencerates were 7.5 W/m2 and 1.6 kW/m2, re-spectively.