Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan.
Proc Natl Acad Sci U S A. 1984 Aug;81(15):4809-13. doi: 10.1073/pnas.81.15.4809.
As an approach to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in greening pumpkin cotyledons, catalase molecules were purified from the two different types of microbody and their structural properties were compared. The purified glyoxysomal catalase was found to consist of four identical subunits (55 kDa), whereas the leaf peroxisomal catalase contains two different forms of monomeric subunit (55 and 59 kDa). These different catalase species cross-reacted with the rabbit antibody raised against the glyoxysomal enzyme. During gel filtration on an Ultrogel AcA 34 column, the leaf peroxisomal 55-kDa polypeptide eluted slightly faster than the leaf peroxisomal 59-kDa polypeptide. The profile of catalase activities exactly paralleled the elution pattern of the 55-kDa molecules, which indicated that the 59-kDa polypeptide was enzymically inactive. Peptide mapping analysis using Staphylococcus aureus protease V8 showed that the glyoxysomal 55-kDa polypeptide was identical to the leaf peroxisomal 55-kDa species, whereas the leaf peroxisomal 59-kDa polypeptide had a different primary structure from the 55-kDa polypeptide. In an in vitro translation system directed by mRNA isolated from etiolated and green cotyledons, glyoxysomal and leaf peroxisomal catalases were synthesized as the identical 59-kDa polypeptide. From peptide mapping analysis, the in vitro-translated 59-kDa polypeptide was found to have a nearly identical primary structure to that of the leaf peroxisomal 59-kDa species. In vivo pulse-chase labeling experiments using etiolated cotyledons showed the conversion of the 59-kDa polypeptide to the 55-kDa molecular species. The overall results strongly indicate that the 59-kDa polypeptide is a precursor form of catalase in pumpkin cotyledons.
作为研究微体转化（乙醛酸体到叶过氧化物体）机制的一种方法，我们从两种不同类型的微体中纯化了过氧化氢酶分子，并比较了它们的结构特性。纯化的乙醛酸体过氧化氢酶由四个相同的亚基（55 kDa）组成，而叶过氧化物体过氧化氢酶则含有两种不同形式的单体亚基（55 和 59 kDa）。这些不同的过氧化氢酶与兔抗乙醛酸体酶抗体发生交叉反应。在 Ultrogel AcA 34 柱上进行凝胶过滤时，叶过氧化物体 55 kDa 多肽的洗脱速度略快于叶过氧化物体 59 kDa 多肽。过氧化氢酶活性的图谱与 55 kDa 分子的洗脱模式完全平行，这表明 59 kDa 多肽在酶学上是无活性的。使用金黄色葡萄球菌蛋白酶 V8 进行肽图谱分析表明，乙醛酸体 55 kDa 多肽与叶过氧化物体 55 kDa 物种完全相同，而叶过氧化物体 59 kDa 多肽的一级结构与 55 kDa 多肽不同。在由黄化和绿色子叶分离的 mRNA 指导的体外翻译系统中，合成了与乙醛酸体和叶过氧化物体过氧化氢酶相同的 59 kDa 多肽。通过肽图谱分析，发现体外翻译的 59 kDa 多肽与叶过氧化物体 59 kDa 物种具有几乎相同的一级结构。在使用黄化子叶的体内脉冲追踪标记实验中，观察到 59 kDa 多肽向 55 kDa 分子物种的转化。总体结果强烈表明，59 kDa 多肽是南瓜子叶中过氧化氢酶的前体形式。
