Vierstra R D, Quail P H
Department of Botany, 139 Birge Hall, University of Wisconsin-Madison, Madison, Wisconsin 53706.
Plant Physiol. 1985 Apr;77(4):990-8. doi: 10.1104/pp.77.4.990.
A spectral, immunochemical, and proteolytic characterization of native 120-kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified ( approximately 200-fold) in the phytochrome-far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr lambda(max) at 730 nanometers, a spectral change ratio (DeltaA(r)/DeltaA(fr)) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH(2) terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH(2) terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr lambda(max) to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH-terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.
本文介绍了来自西葫芦（Cucurbita pepo L.）的天然120千道尔顿（kD）光敏色素的光谱、免疫化学和蛋白水解特性，并与先前报道的来自燕麦（Avena sativa）的天然124-kD光敏色素进行了比较。在蛋白酶抑制剂苯甲基磺酰氟存在的情况下，采用最初用于纯化124-kD燕麦光敏色素的方法的改进方法，以光敏色素远红光吸收形式（Pfr）对该分子进行了部分纯化（约200倍）。所获得制剂的光谱特性与124-kD燕麦光敏色素所描述的特性无法区分，包括在730纳米处的Pfr λ（max）、光谱变化率（ΔA（r）/ΔA（fr））为1.05，以及在有无连二亚硫酸钠的情况下Pfr向红光吸收形式（Pr）的暗逆转可忽略不计。体外缺乏暗逆转与西葫芦光敏色素（与大多数其他双子叶植物的光敏色素一样）在体内表现出大量暗逆转的观察结果形成对比。用多克隆抗体进行的Ouchterlony双向免疫扩散分析表明，120-kD西葫芦光敏色素与124-kD燕麦光敏色素在免疫上不同。然而，尽管存在这种差异，但对蛋白水解消化物的免疫印迹分析已鉴定出至少三个两种光敏色素共有的空间上分开的表位。利用内源性蛋白酶构建了西葫芦光敏色素的肽图，并研究了特定结构域在光感受器整体结构中所起的作用。靠近NH（2）末端的一个结构域对分子的光谱完整性至关重要，这表明该结构域起的结构作用类似于燕麦光敏色素NH（2）末端附近的一个结构域。该结构域的蛋白水解去除优先发生在Pr中，其去除会使Pfr λ（max）移至722纳米，将光谱变化率提高到1.3，并显著提高暗逆转率。该结构域在进化上不同的植物物种中的明显保守性及其在光转化时参与构象变化，使其可能与光敏色素作用机制相关。凝胶过滤研究的初步证据表明，多肽的55-kD无发色团COOH末端区域包含一个负责光敏色素单体二聚化的结构域。
