Molecular Mechanisms Underlying Differentiation of Microbodies.

采用免疫化学方法，从绿南瓜子叶聚(A)^+ -RNA构建的cDNA表达文库中分离到一个叶片过氧化物酶glycolate oxidase （E C 1.1.3.1）的cDNA克隆。Northern blot分析表明，该探针可与南瓜子叶中一个1.5 kb的mRNA进行杂交，杂交mRNA的量在子叶变绿过程中显著增加；免疫印迹分析表明，暗生子叶中乙醇酸氧化酶的量较低，在南瓜子叶变绿过程中增加。这一观察结果表明，在子叶变绿过程中，叶片过氧化物酶（乙醇酸氧化酶）的诱导是由于mRNA水平的增加。南瓜子叶变绿过程中，糖酶体向叶片过氧化物酶体的功能转变后，衰老过程中，叶片过氧化物酶体向糖酶体的反向微体转变发生。用蛋白a -金免疫细胞化学标记分析了反微体转化，使用了抗叶过氧化物酶、乙醇酸氧化酶和抗两种gloxysomal酶（即苹果酸合成酶和异柠檬酸裂解酶）的抗体。在衰老过程中，微体中乙醇酸氧化酶的标记强度降低，而苹果酸合成酶和异柠檬酸裂解酶的标记强度显著增加。不同大小的蛋白a -金颗粒双标记实验表明，衰老南瓜子叶微体中存在叶片过氧化物酶体和乙氧化酶体，说明衰老过程中叶片过氧化物酶体直接转化为乙氧化酶体。

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.