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Biosynthesis of rat liver peroxisomal serine:pyruvate aminotransferase.

大鼠肝脏过氧化物酶体丝氨酸的生物合成：丙酮酸转氨酶。

基本信息

批准号：
62570104
负责人：
ICHIYAMA Arata
金额：
$ 1.41万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

In rat liver, there are two types of serine:pyruvate aminotransferase (SPT) as to the organelle distribution. One is a mitochondrial enzyme (SPTm) and the other a peroxisomal enzyme (SPTP). The properties of the two enzymes are apparently indistinguishable, but administration of glucagon or insulin causes the selective induction of SPTm. We have shown previously that SPTm is biosynthesized from 1900 nt-mRNA as 45 kDa-precursor which is then specifically translocated into mitochondria and converted therein to mature SPTm. In this study, the precesses involved in the biosynthesis of SPTp were investigated. Results obtained are as follows: 1. On RNA blot analysis, 1700 nt-mRNA was detected in addition to 1900 nt-mRMA. The structure of the two mRNAs were extremely similar except that 1700 nt-mRNA lacks about 70 nucleotides of 5'-terminal sequence of 1900 nt-mRNA and has about 100 nucl eotides shorter poly-A tail when compared with 1900 nt-mRNA formed shortly after the administration of the hormones.2. Southern blot analysis of rat genomic DNA showed that the SPT gene is single. The analyses on primer extension and S1 nuclease mapping using DNA fragment of the genomic clone revealed that 1900 nt- and 1700 nt-mRNAs are transcribed from different initiation sites in the same exon 1. 3. In cell-free translation, 43 kDa-product was detected in addition to 45 kDa-product. the 43 KDa-product was suggested to be related to SPTp bv a similar response to hormones or peroxisome proliferators. However, the 43 kDa-product was bound to but not taken up into peroxisomes in vitro under conditions so far tested. 4. The N-terminal sequence of SPTm was determined to be Met-Gly-Ser-His---. The N-terminal Met corresponded to the initiation Met in the translation of 1700 nt-mRNA, suggesting that SPTm and SPTp share the same primary structure, although their biosynthetic processes and hence the organelle Colalization differ.

在大鼠肝脏中，有两种类型的丝氨酸：丙酮酸转氨酶（SPT）。一种是线粒体酶（SPTm），另一种是过氧化物酶（SPTP）。这两种酶的性质显然难以区分，但胰高血糖素或胰岛素的施用可选择性诱导SPTm。我们之前已经证明，SPTm是由1900 nt-mRNA作为45 kda前体生物合成的，然后特异性地转位到线粒体中并在其中转化为成熟的SPTm。本研究对SPTp的生物合成过程进行了研究。实验结果如下：1。在RNA印迹分析中，除了1900 nt-mRMA外，还检测到1700 nt-mRNA。两种mrna的结构非常相似，除了1700 nt-mRNA比1900 nt-mRNA缺少约70个核苷酸的5'端序列，并且与1900 nt-mRNA相比，在给药后不久形成的poly-A尾部短了约100个核苷酸。大鼠基因组DNA的Southern blot分析表明，SPT基因为单基因。利用基因组克隆DNA片段进行引物延伸和S1核酸酶定位分析，发现在同一外显子1的不同起始位点分别转录了1900个nt-和1700个nt- mrna。3. 在无细胞翻译中，除了检测到45 kda产物外，还检测到43 kda产物。通过对激素或过氧化物酶体增殖物的类似反应，表明43 kda产物与SPTp有关。然而，在目前测试的条件下，43 kda产品在体外与过氧化物酶体结合，但没有被吸收。4. 确定SPTm的n端序列为Met-Gly-Ser-His——。n端Met对应于1700 nt-mRNA翻译中的起始Met，这表明SPTm和SPTp具有相同的初级结构，尽管它们的生物合成过程和细胞器共化不同。