Molecular Mechanisms Underlying Differentiation of Microbodies.

用免疫化学方法从绿色南瓜子叶poly（A）^+ -RNA构建的cDNA表达文库中克隆了一个叶过氧化物酶-乙醇酸氧化酶的cDNA克隆（EC 1.1.3.1）。北方印迹分析表明，该探针可与南瓜子叶中的一个1.5kb的mRNA杂交，且在子叶变绿过程中，可杂交的mRNA的量显著增加。免疫印迹分析表明，乙醇酸氧化酶在黑暗生长的子叶中含量较低，而在南瓜子叶变绿过程中含量增加。这一观察结果表明，诱导叶片过氧化物酶，乙醇酸氧化酶，在子叶的绿化过程中是由于mRNA水平的增加。在南瓜子叶的绿化过程中的功能转换后，叶片过氧化物酶体的功能转换到叶片过氧化物酶体，叶片过氧化物酶体的逆转微体转换到乙醛酸酶体在衰老过程中发生。用蛋白质A-金免疫细胞化学标记进行分析的反向微体转换使用抗体对叶过氧化物酶体酶，乙醇酸氧化酶，和ageinst两个粘体酶，即苹果酸合酶和异柠檬酸裂解酶。乙醇酸氧化酶的标记强度下降，而在苹果酸合酶和异柠檬酸裂解酶的情况下，强度显着增加衰老过程中的微体。用不同大小的蛋白A-金颗粒进行双标记实验，结果表明，叶片过氧化物酶体酶和乙二醛酶体酶同时存在于衰老过程中的南瓜子叶微体中，表明叶片过氧化物酶体在衰老过程中直接转化为乙二醛酶体。

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.