The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.
The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.
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DOI：
10.1016/s0021-9258(19)67859-2
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发表时间：
1991-02
期刊：
影响因子：
--
通讯作者：
T. Sato;P. Oeller;A. Theologis
中科院分区：
文献类型：
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作者：
T. Sato;P. Oeller;A. Theologis
植物激素乙烯生物合成途径中的关键调节酶是1-氨基环丙烷-1-羧酸合成酶(EC 4.4.1.14)。我们用吲哚乙酸+苄基腺嘌呤+氨基氧乙酸+氯化锂处理南瓜果实组织，将ACC合成酶部分纯化了6000倍。该酶的比活力为35000nmol/h/mg蛋白质，最适pH为9.5，等电点为5.0，对S-腺苷蛋氨酸的Km为17微米，是由两个相同的亚基组成的二聚体，每个亚基约46000Da。该亚基在体内以55,000-Da的形式存在，其大小与主要的体外翻译产物相似。序列分析表明，Acc合成酶基因的编码区由493个氨基酸组成，相当于一个55,779-Da的多肽，该编码序列在大肠杆菌中以β-半乳糖苷酶的羧基末端杂合或非杂合多肽的形式表达，催化S-腺苷甲硫氨酸转化为Acc(Sato，T.，and Theologis，A.(1989)Proc.娜塔莉。阿卡德。SCI。美国86,6621-6625)。免疫印迹实验表明，β-半乳糖苷酶杂化多肽的分子质量为17万Da，最大的非杂化多肽的大小为53000Da。这些数据表明，该酶在蛋白质纯化过程中是翻译后加工的。
The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.