Cloning and expression of dihydrolipoamide acyl-transferase gene and localization of its product in mitochondria

1.猪心二氢硫酰胺乙酰和琥珀酰基转移酶(LAT，LST)分别是丙酮酸脱氢酶复合体(PDC)和2-氧戊二酸脱氢酶复合体(OGDC)的核心酶，用单一高效液相色谱法从PDC和OGDC中分离得到，含有共价结合的硫辛酸作为辅基。这两种酶都没有表现出器官特异性。在兔体内制备了两种酶的抗血清，并与人的酶表现出较强的交叉反应。用免疫细胞化学技术检测LST在线粒体内的分布，发现LST在基质中沿突起呈金黄色分布。用异硫氰酸胍萃取法从一天龄的猪心脏中分离出Poly(A)^+RNA。免疫沉淀法从S标记的无细胞翻译产物中检测LAT(80 KDa)和LST(50 KDa)前体。在~lt；lambda&gt；gtll载体中构建了全长600bp~2kb的文库。这两种前体进入大鼠线粒体的转运是在…更多的mpt。用免疫筛选法从HeLa细胞中分离到人LAT和LST基因片段，用噬菌体cDNA克隆杂交法从人包皮成纤维细胞质粒库中分离到几乎全长的cDNAs。核苷酸序列分析表明，LAT插入了2.3kb，LST插入了2.9kb。从人胎盘cDNA文库中分离出阳性噬菌体克隆，序列分析显示LAT插入1.9kb，LST插入2.8kb。测定了猪LAT(38个残基)和LST(32个残基)的NH_2末端氨基酸序列和两种酶(5个残基)的COOH末端氨基酸序列，并与由核苷酸序列推导出的人类酶的氨基酸序列进行了比较。预测的两种酶的序列在两个空间之间的同源性较差。利用克隆的人LAT和LST基因，从EMBL4中的人白细胞基因组DNA文库中筛选出两种酶的基因组DNA。较少

1. Porcine heart dihydrolipoamide acetyl- and succinyl-transferase(LAT, LST), which are a core enzyme of pyruvate and 2-oxoglutarate dehydrogenase complexes (PDC, OgDC), respectively, were isolated from PDC and OGDC by a single HPLC, and contained covalently bound-lipoic acid as prosthetic group. Both enzymes did not exhibit organ organ specificities.2. Antisera against two enzymes were raised in rabbit and showed high cross-reactivities with human enzymes. Intra-mitochondrial distribution of LST was examined by an immunocytochemical technique and the gold particles representing LST were found in the matrix along crista.3. Poly (A)^+ RNAs were isolated from a day-old porcine heart by guanidine isothiocyanate extraction. The precursors of LAT (80KDa) and LST (50KDa) were detected by immunoprecipitation from an ^<35>S-labeled cell-free translation products. The cDNA (600bp - 2kb) library in <lambda>gtll vector were prepared. Transport of the two precursors into rat mitochondria were atte … More mpted.4. Human LAT and LST cDNA fragments were isolated from HeLa cell <lambda>gtll cDNA library by immunoscreening and the almost full-length cDNAs were isolated from human foreskin fibroblast plasmid library by colony hybridization with phage cDNAs. Nucleotide sequence analyses of plasmid clones revealed an insert of 2.3kb for LAT and one of 2.9kb for LST, respectively. From human placenta cDNA library positive phage clones were isolated and sequence analyses revealed an insert of 1.9kb for LAT and one of 2.8kb for LST, respectively.5. Mino acid sequences of NH_2-termini of porcine LAT (38 residues) and LST (32 residues) and those of COOH-termini of Two enzymes (5 residues) were determined and compared with those of human enzymes deduced from the nucleotide sequences. The homologies of the predicted sequences of two enzymes among two spacies were poor.6. By using cloned cDNAs for human LAT and LST the screening of genomic dNAS for two enzymes are attempled from human leucocyte genomic DNA library in EMBL4. Less