Roberts L M, Lord J M
Eur J Biochem. 1981 Sep;119(1):43-9. doi: 10.1111/j.1432-1033.1981.tb05574.x.
Total polyadenylated RNA isolated from 3-day-old germinating castor bean endosperm was translated in the rabbit reticulocyte lyase cell-free protein-synthesizing system in either the presence or absence of canine pancreatic microsomal vesicles. Isocitrate lyase was immunoprecipitated from the translational products using rabbit antibodies raised against the purified glyoxysomal enzyme. Isocitrate lyase synthesized in vitro had the same apparent molecular weight as the authentic glyoxysomal enzyme irrespective of whether synthesis occurred in the presence or absence of microsomes. Furthermore, in contrast to several proteins encoded by mRNA isolated from maturing seed endosperm tissue, isocitrate lyase was not cotranslationally segregated into the lumen of the microsomal vesicles. Intact 3-day-old endosperm tissue was labelled in vivo by adding [35S]methionine and at various times the tissue was homogenized and the cellular organelles fractionated. Separated fractions were assayed for newly synthesized isocitrate lyase. Immunoreactive product was initially detected in the cytosolic fraction and subsequently began to accumulate in glyoxysomes. The results indicate that glyoxysomal isocitrate lyase is synthesized on free polysomes and released into the cytosol. Transfer to the matrix of the glyoxysome involves a posttranslational membrane translocation step which is not accompanied by proteolytic cleavage of the polypeptide chain.
从3日龄萌发的蓖麻籽胚乳中分离得到的总多聚腺苷酸化RNA，在有无犬胰腺微粒体囊泡存在的情况下，于兔网织红细胞裂解物无细胞蛋白质合成系统中进行翻译。使用针对纯化的乙醛酸循环体酶产生的兔抗体，从翻译产物中免疫沉淀异柠檬酸裂解酶。无论翻译是在有或无微粒体的情况下进行，体外合成的异柠檬酸裂解酶与真正的乙醛酸循环体酶具有相同的表观分子量。此外，与从成熟种子胚乳组织分离的mRNA编码的几种蛋白质不同，异柠檬酸裂解酶不会共翻译分隔到微粒体囊泡腔中。通过添加[35S]甲硫氨酸对完整的3日龄胚乳组织进行体内标记，并在不同时间将组织匀浆并对细胞器进行分级分离。对分离的级分进行新合成的异柠檬酸裂解酶检测。免疫反应性产物最初在细胞质级分中检测到，随后开始在乙醛酸循环体中积累。结果表明，乙醛酸循环体异柠檬酸裂解酶在游离多核糖体上合成并释放到细胞质中。转移到乙醛酸循环体基质涉及翻译后膜易位步骤，该步骤不伴随多肽链的蛋白水解切割。
