He P M, Zhang R X, Zhang D B, Zhao J H, Liang W Q
Agriculture University of Nanjing, Nanjing 210095.
Shi Yan Sheng Wu Xue Bao. 2001 Mar;34(1):18-23.
Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalization of Rubisco of Chlorella spp. 640909, and the Native-PAGE and, SDS-PAGE and Western bloting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothecoides, The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55 KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10 nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae.
1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）是植物细胞光合作用卡尔文循环中的首个关键酶。本文主要研究了小球藻640909中Rubisco的金免疫定位、非变性聚丙烯酰胺凝胶电泳（Native-PAGE）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析，以及对蛋白核超微结构的观察。非变性聚丙烯酰胺凝胶电泳结果显示有一条主带，用来自原球藻的Rubisco抗体进行蛋白质免疫印迹证明其为Rubisco条带；小球藻640909的Rubisco与原球藻Rubisco大亚基抗体的特异性免疫反应表明，这两种小球藻中Rubisco的大亚基蛋白具有高度同源性。SDS-PAGE和蛋白质免疫印迹图谱显示，小球藻640909中Rubisco大亚基的分子量约为55千道尔顿。电子显微镜下蛋白核超微结构呈长方形，嵌入淀粉鞘中，并在蛋白核中央纵向通道贯穿2个肿胀的类囊体。蛋白核与叶绿体基质之间存在一些连接。通过用大亚基和全酶抗体包埋切片，然后用与10纳米金颗粒偶联的羊抗兔免疫球蛋白G（Sigma产品）作为二抗，通过免疫电子显微镜研究了小球藻640909叶绿体中Rubisco大亚基和全酶的分布情况；结果显示，大亚基和全酶抗体均对蛋白核以及淀粉鞘区域进行了大量标记，而质体的类囊体区域标记较轻；并且全酶抗体对蛋白核表面的标记比重链抗体更重。这表明蛋白核和淀粉鞘具有光合作用功能。集中在蛋白核和淀粉鞘中的Rubisco对于藻类光合作用固定二氧化碳具有重要作用。
