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

1.分别从猪心丙酮酸脱氢酶复合物（PDC）和2-酮戊二酸脱氢酶复合物（OgDC）中分离得到了含有共价结合的硫辛酸辅基的猪心二氢硫辛酰胺乙酰基和琥珀酰转移酶（LAT，LST）。两种酶均未表现出器官特异性.抗血清对这两种酶提出了在兔和人的酶表现出高度的交叉反应性。用免疫细胞化学技术检测LST在线粒体内的分布，并发现代表LST的金颗粒沿沿着分布于基质中.通过异硫氰酸胍提取法从一日龄猪心中分离出Poly（A）^+ RNA。用免疫沉淀法检测LAT（80 KDa）和LST（50 KDa）的前体<35>。制备了gt 11载体中的cDNA文库（600 bp- 2kb）<lambda>。两种前体物质进入大鼠线粒体的转运被证实， ...更多信息 mpted.4.通过免疫筛选从HeLa细胞gtll cDNA库中分离出人LAT和LST cDNA片段<lambda>，并通过与噬菌体cDNA的菌落杂交从人包皮成纤维细胞质粒库中分离出几乎全长的cDNA。质粒克隆的核苷酸序列分析显示LAT和LST分别插入2.3kb和2.9kb。从人胎盘cDNA文库中筛选出阳性噬菌体克隆，序列分析显示分别有一个1.9kb的LAT和一个2.8kb的LST插入片段.测定了猪LAT（38个氨基酸残基）和LST（32个氨基酸残基）的NH_2-端氨基酸序列及两种酶的COOH-端氨基酸序列（5个氨基酸残基），并与人的相应序列进行了比较。两种酶的预测序列在两个空间的同源性较差.利用克隆的人LAT和LST的cDNA，从EMBL 4中的人白细胞基因组DNA文库中筛选两种酶的基因组dNAS。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