Biosynthesis of rat liver peroxisomal serine:pyruvate aminotransferase.

在大鼠肝脏中，丝氨酸：丙酮酸氨基转移酶（SPT）的细胞器分布有两种类型。一种是线粒体酶（SPTm），另一种是过氧化物酶体酶（SPTP）。这两种酶的性质显然是不可区分的，但胰高血糖素或胰岛素的施用导致SPTm的选择性诱导。我们先前已经表明，SPTm是从1900 nt-mRNA作为45 kDa-前体生物合成的，然后特异性地易位到线粒体中并在其中转化为成熟的SPTm。本研究对SPTp生物合成过程进行了研究。主要研究结果如下：1.在RNA印迹分析中，除了1900 nt-mRMA之外，还检测到1700 nt-mRNA。两种mRNA的结构非常相似，只是1700 nt-mRNA的5 '端序列比1900 nt-mRNA少了约70个核苷酸，而且与1900 nt-mRNA相比，多聚腺苷酸尾短了约100个核苷酸. Southern杂交分析表明，SPT基因为单基因。引物延伸分析和S1核酸酶图谱分析表明，1900 nt和1700 nt的mRNA是从同一外显子1的不同起始位点转录的。3.在无细胞翻译中，除了45 kDa产物外，还检测到43 kDa产物。43 kDa产物被认为与SPTp有关，对激素或过氧化物酶体增殖物有类似的反应。然而，在迄今为止测试的条件下，43 kDa产物在体外与过氧化物酶体结合但不被其吸收。4. SPTm的N端序列为Met-Gly-Ser-His--。N-末端Met对应于1700 nt-mRNA翻译中的起始Met，表明SPTm和SPTp共享相同的一级结构，尽管它们的生物合成过程以及因此细胞器Colalization不同。

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.