【作者】 郭晋雅；

【导师】 徐杰；

【作者基本信息】 华南师范大学 ， 植物学， 2007， 硕士

【摘要】 乙醇酸氧化酶(EC 1．1．3．15，GO)是植物光呼吸的关键酶，由一种分子量(Mr)为40kD的亚基构成。针对前人对GO研究发现的GO单一分子结构与多个等电点(pI)之间的矛盾，本实验组在前期工作中已初步证实GO可以与一种富含苯丙氨酸的蛋白结合形成GO复合蛋白。本文在此基础上以免疫杂交为主要手段，对GO复合蛋白的组成、GO与富含苯丙氨酸蛋白的相互关系、富含苯丙氨酸蛋白的性质及其对GO电泳行为的影响进行了研究，主要结果如下：通过10％醋酸沉淀、硫酸铵分级沉淀、Sephadex G-50分子筛柱层析、DEAE-cellulose-52离子交换柱层析，从菜心(Brassica parachinensis Bailey)绿叶中获得具有GO活性、pI＞8.3的GO复合蛋白。该GO复合蛋白在SDS-PAGE中呈Mr为40kD的单带。经醋酸纤维素薄膜电泳分析表明，GO复合蛋白中含有在SDS变性下向负极泳动的蛋白成分。氨基酸组成测定的结果表明，向负极泳动的蛋白富含苯丙氨酸，其含量达12.96％。GO复合蛋白经SDS变性后，进行Sephadex G-100凝胶过滤得到两种蛋白组分：GC-Ⅰ和GC-Ⅱ。凝胶过滤曲线表明GC-Ⅰ的Mr约为67kD；GC-Ⅱ的Mr介于14kD和67kD之间。而GC-Ⅰ经SDS-PAGE后出现Mr分别为40kD和14kD的两条带，或只出现Mr为40kD的一条带；GC-Ⅱ在SDS-PAGE中则出现Mr为14kD的一条带，或者未出现任何条带。该结果表明Mr为14kD的蛋白与GO有密切联系。用GO 40kD亚基抗体和GO复合蛋白抗体对GO复合蛋白作斑点杂交和SDS-PAGE Western-blot分析，发现GO 40kD亚基抗体可以识别非SDS变性的GO复合蛋白，而GO复合蛋白抗体对SDS变性下GO的40kD亚基识别能力很弱。据此推测：富含苯丙氨酸的蛋白主要位于GO复合蛋白的外部，而GO主要位于内部；富含苯丙氨酸的蛋白不完全包被GO，其掩盖了GO的抗原决定簇。用GO复合蛋白抗体对菜心绿叶粗蛋白作SDS-PAGE Western-blot分析，杂交主带为54kD和14kD，表明粗蛋白中54kD和14kD的蛋白与GO复合蛋白有免疫源性。从粗蛋白中回收上述54kD和14kD的蛋白，用GO复合蛋白抗体进行SDS-PAGE Western-blot分析，膜上出现Mr从30kD～97kD的一系列阶梯状杂交条带，条带密集处位于97kD和54kD附近。该结果说明GO复合蛋白与SDS的结合比例是可变的，在SDS-PAGE中呈现出多种迁移率。制备菜心绿叶粗蛋白中14kD蛋白的抗体，用该抗体对GO复合蛋白进行斑点杂交和SDS-PAGE Western-blot分析，发现14kD蛋白抗体可以识别非SDS变性的GO复合蛋白，但不能识别SDS变性下GO的40kD亚基，表明Mr为14kD的蛋白可能是GO复合蛋白中富含苯丙氨酸的蛋白。用GO 40kD亚基抗体、GO复合蛋白抗体、14kD蛋白抗体对GO复合蛋白进行SDS-醋酸纤维素薄膜免疫杂交分析，发现GO复合蛋白经SDS变性处理后，其中GO同时向正极和负极泳动，而富含苯丙氨酸的蛋白主要向负极泳动。更多还原

【Abstract】 Glycolate oxidase （EC 1.1.3.15, GO） is a peroxisomal flavin-dependent enzymein the photorespiratory pathway in plants, which is consisted of identical subunits ofMr 40kD. It was confirmed that GO was bound with a phenylalanine-rich proteinforming a protein complex which was named GO complex. In this paper, therelationship between GO and the phenylalanine-rich protein in GO complex wasstudied.GO complex was separated from green leaves of Brassica parachininensisBailey using 10% acetic acid precipitation, （NH₄）₂SO₄ precipitation, Sephadex G-50molecular sieve chromatograph and DEAE-Cellulose negative ion-exchange chromat-ograph. GO complex exhibited one band of Mr 40kD in SDS-PAGE and GO enzymeactivity was determined in GO complex. Proteins in GO complex migrated to cathodewas detected by SDS-acetate cellulose membrane eletrophoresis. The component ofamino acid in the proteins migrated to cathode in SDS-PAGE was assayed. Thecontent of phenylalanine of proteins in cathode was 12.96%.GC-Ⅰprotein and GC-Ⅱprotein were obtained when SDS-denatured GOcomplex was subjected to Sephadex G-100 gel filtration chromatography. Molecularweight of GC-Ⅰprotein and GC-Ⅱprotein are 67 kD and between 14 kD and 67 kDrespectively. But in SDS-PAGE GC-Ⅰprotein exhibited two bands of Mr 40 kD and14 kD or one band of Mr 40 kD, while GC-Ⅱprotein exhibited one band of Mr 14 kDor no band. This result indicated that the protein of Mr 14kD related closely to GO.GO complex was subjected to dot blot and SDS-PAGE Western blot usingantibody against GO 40 kD subunit and antibody against GO complex respectively,which showed that the GO complex was recognized by antibody against GO 40 kDand GO 40 kD subunit wasn’t recognized by antibody against GO complex. The resultsuggested that the phenylalanine-rich protein was in the outside of GO complex andGO was in the inside of GO complex.Crude extract from green leaves of Brassica parachininensis Bailey wassubjected to SDS-PAGE Western blot using antibody against GO complex and there were two main immunity bands of Mr 54 kD and Mr 14 kD. Extract the 54 kD and 14kD protein of Crude extract from green leaves of Brassica parachininensis Bailey,using the antibody against GO complex to SDS-PAGE Western blot assay. There werea set of ladder bands varied from Mr 30 kD to 97 kD, Which showed that the quantityof SDS bound by GO complex was variable.Prepared the antibody against 14kD protein of crude extract from green leavesof Brassica parachininensis Bailey. This antibody was used to dot blot andSDS-PAGE Western blot assay of GO complex., which showed that the antibodyagainst 14 kD protein recognized the GO complex and didn’t recognize the 40 kDsubunit of GO. The results indicated that 14kD protein was the phenylalanine-richprotein in GO complex.GO complex was subjected to SDS-acetate cellulose membrane immuno-hybridization using the antibody against GO complex, the antibody against GO 40kD subunit and the antibody against 14kD protein respectively. The results suggestedthat GO migrated to anode and cathode, and the phenylalanine-rich protein migratedto cathode.更多还原