Mori H, Takeda-Yoshikawa Y, Hara-Nishimura I, Nishimura M
Department of Regulation Biology, National Institute for Basic Biology, Okazaki, Japan.
Eur J Biochem. 1991 Apr 23;197(2):331-6. doi: 10.1111/j.1432-1033.1991.tb15915.x.
A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.
通过对由黄化南瓜子叶富含聚腺苷酸的RNA构建的cDNA表达文库进行免疫筛选，鉴定出一个编码乙醛酸循环体苹果酸合酶（EC 4.1.3.2）的cDNA克隆。对这个1979个核苷酸的cDNA的DNA序列测定揭示了一个1698个核苷酸的开放阅读框，其编码一个64632道尔顿的多肽。通过对纯化酶经酸处理产生的片段进行肽序列分析获得的三个序列进行匹配，证实了苹果酸合酶cDNA的鉴定。Northern印迹分析表明，该探针与来自黄化南瓜子叶的一种单一的2.3 kb mRNA杂交，而绿色南瓜子叶中不存在这种mRNA。在推导的氨基酸序列比较中，发现南瓜苹果酸合酶与来自油菜和大肠杆菌的苹果酸合酶分别具有83%和48%的相似性。基于这三种苹果酸合酶的氨基酸序列相似性和亲水图谱，讨论了将该酶靶向微体的信号。
