Molecular Mechanisms Underlying Differentiation of Microbodies.

微生物分化的分子机制。

基本信息

批准号：
04454017
负责人：
NISHIMURA Mikio
金额：
$ 4.48万
依托单位：
National Institute for Basic Biology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

A cDNA clone for a leaf-peroxisomal enzyme, glycolate oxidase (E C 1.1.3.1), was isolated by an immunochemical method from a cDNA expression library constructed from poly (A)^+ -RNA of green pumpkin cotyledons. Northern blot analysis showed that the probe could hybridizable with a single 1.5-kb species of mRNA from pumpkin cotyledons and that the amount of the hybridizable mRNA increased dramatically during greening of the cotyledons, Immunoblot analysis showed that the amount of glycolate oxidase was low in darkgrown cotyledons and increased during greening of pumpkin cotyledons. This observation indicates that the induction of a leaf peroxisomal enzyme, glycolate oxidase, during greening of the cotyledons is due to an increase in level of the mRNA.After the function transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and ageinst two gloxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increase strikingly. Double labeling experments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomal are directly transformed to glyoxysomes during, senescence.

通过免疫化学方法从绿色南瓜子叶的聚(A)^+-RNA构建的cDNA表达文库中分离叶过氧化物酶体酶、乙醇酸氧化酶(EC 1.1.3.1)的cDNA克隆。 Northern印迹分析表明该探针可以与南瓜子叶中单个1.5-kb的mRNA杂交，并且在子叶变绿过程中可杂交的mRNA量急剧增加。免疫印迹分析显示暗生南瓜中乙醇酸氧化酶的量较低子叶，并在南瓜子叶变绿期间增加。这一观察结果表明，在子叶变绿过程中，叶过氧化物酶体酶（乙醇酸氧化酶）的诱导是由于 mRNA 水平的增加。在南瓜子叶变绿过程中，乙醛酸酶体功能转变为叶过氧化物酶体后，反向微体叶子过氧化物酶体向乙醛酸酶体的转变发生在衰老过程中。使用针对叶过氧化物酶体酶、乙醇酸氧化酶和抗两种胶质酶体酶（即苹果酸合酶和异柠檬酸裂合酶）的抗体进行用蛋白A-金的免疫细胞化学标记来分析反向微体转变。在衰老过程中，微生物中乙醇酸氧化酶的标记强度下降，而苹果酸合酶和异柠檬酸裂解酶的标记强度则显着增加。不同大小的蛋白A-金颗粒双标记实验表明，叶过氧化物酶体酶和乙醛酸酶体共存于衰老南瓜子叶的微体中，表明叶过氧化物酶体酶在衰老过程中直接转化为乙醛酸体。