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

1. 猪心脏二氢脂酰胺乙酰转移酶（LAT）和琥珀酰转移酶（LST）分别是丙酮酸和2-氧戊二酸脱氢酶复合物（PDC, OgDC）的核心酶，用高效液相色谱法从PDC和OgDC中分离得到，并以共价结合的硫辛酸为假基。两种酶均未表现出器官特异性。两种酶的抗血清在家兔体内培养，并与人体内的酶具有较高的交叉反应性。利用免疫细胞化学技术检测LST在线粒体内的分布，沿嵴在基质中发现了代表LST的金颗粒。采用胍异硫氰酸酯萃取法从日龄猪心脏中分离到Poly (A)^+ rna。通过免疫沉淀法检测LAT （80KDa）和LST （50KDa）的前体，这些前体来自于^<35> s标记的无细胞翻译产物。制备了<lambda>gtll载体的cDNA （600bp - 2kb）文库。这两种前体在大鼠线粒体中的转运被证实。通过免疫筛选从HeLa细胞<lambda>gtll cDNA文库中分离到人LAT和LST cDNA片段，并与噬菌体cDNA进行集落杂交从人包皮成纤维质粒文库中分离到几乎全长的cDNA片段。质粒克隆的核苷酸序列分析显示，LAT和LST分别有2.3kb和2.9kb的插入。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和LST分别插入1.9kb和2.8kb。测定了猪LAT（38个碱基）和LST（32个碱基）的nh_2末端的氨基酸序列，以及2种酶（5个碱基）的cooh末端的氨基酸序列，并与人类酶的核苷酸序列进行了比较。两种酶在两个空间间的预测序列同源性较差。利用克隆的人LAT和LST基因组DNA，从人白细胞基因组DNA文库中筛选了两种酶的基因组DNA。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