Kidd D G, Lagarias J C
Department of Biochemistry and Biophysics, University of California, Davis 95616.
J Biol Chem. 1990 Apr 25;265(12):7029-35.
A method to purify the phytochrome photoreceptor from the unicellular green alga Mesotaenium caldariorum is presented. Preparative scale formation of algal protoplasts and controlled osmotic cell lysis have permitted separation of intact organelles from the phytochrome-enriched soluble protein fraction. We have utilized the observation that red light-absorbing (Pr) and far-red light-absorbing (Pfr) forms of phytochrome are differentially retained on an anion exchange matrix to purify M. caldariorum phytochrome to apparent homogeneity. M. caldariorum phytochrome preparations with A650/A280 ratios greater than 0.78 exhibit a single 120-kDa band on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Immunoblot analyses using a cross-reactive pea phytochrome monoclonal antibody reveal that 1) the 120-kDa band represents the full-length polypeptide, 2) phytochrome is predominantly localized in the algal cytoplasm, and 3) there are 150,000-250,000 phytochrome molecules/cell. Steric exclusion high pressure liquid chromatography analysis under nondenaturing conditions indicates that M. caldariorum phytochrome has an apparent mass of 355 kDa. The absorption maxima for Pr and Pfr are 650 and 722 nm, respectively. Both are blue-shifted compared with those of phytochromes from dark-grown angiosperm tissue. The molar absorption coefficient for Pr at 650 nm is 86,800 +/- 2800 liter mol-1 cm-1, which is lower than that of higher plant phytochromes. The significance of the similarities and differences of the molecular properties of phytochromes from M. caldariorum and higher plant sources is discussed.
本文介绍了一种从单细胞绿藻嗜热微星藻中纯化光敏色素光感受器的方法。通过制备规模的藻类原生质体形成和可控的渗透细胞裂解，得以从富含光敏色素的可溶性蛋白质组分中分离出完整的细胞器。我们利用了如下观察结果：光敏色素的红光吸收型（Pr）和远红光吸收型（Pfr）在阴离子交换基质上的保留情况不同，从而将嗜热微星藻光敏色素纯化至表观均一性。在银染的十二烷基硫酸钠-聚丙烯酰胺凝胶上，A650/A280比值大于0.78的嗜热微星藻光敏色素制剂呈现出一条单一的120 kDa条带。使用交叉反应性豌豆光敏色素单克隆抗体进行的免疫印迹分析表明：1）120 kDa条带代表全长多肽；2）光敏色素主要定位于藻类细胞质中；3）每个细胞中有150,000 - 250,000个光敏色素分子。非变性条件下的空间排阻高压液相色谱分析表明，嗜热微星藻光敏色素的表观质量为355 kDa。Pr和Pfr的吸收最大值分别为650和722 nm。与来自黑暗生长的被子植物组织的光敏色素相比，二者的吸收峰均发生了蓝移。Pr在650 nm处的摩尔吸收系数为86,800 +/- 2800升·摩尔-1·厘米-1，低于高等植物光敏色素的摩尔吸收系数。本文还讨论了嗜热微星藻和高等植物来源的光敏色素分子特性异同的意义。
