Lau K S, Griffin T A, Hu C W, Chuang D T
Department of Medicine, Veterans Administration Medical Center, Cleveland, Ohio 44106.
Biochemistry. 1988 Mar 22;27(6):1972-81. doi: 10.1021/bi00406a025.
The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods. Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis. The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component. A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe. The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2. A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe. Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively. Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions. In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail. Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase. The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain. Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.
采用蛋白质免疫印迹法、肽序列分析及 cDNA 克隆法，对牛和人支链 α - 酮酸脱氢酶复合体中二氢硫辛酰胺转乙酰基酶（E2）组分的亚基结构及保守性进行了研究。用针对十二烷基硫酸钠（SDS）变性牛 E2 亚基制备的兔抗血清识别 E2 核心内部以及 E2 链的第一个铰链区，但通过蛋白质免疫印迹分析确定，该抗血清与含硫辛酰胺结构域不发生反应。用针对天然 E2 组分的抗体证实了含硫辛酰胺结构域缺乏抗原性。以上述抗天然和抗 SDS 变性 E2 的免疫球蛋白 G 混合物为探针，从 λgt11 载体的人肝 cDNA 文库中分离出一个人 E2 cDNA（1.6 kb）。通过核苷酸测序确定了人 E2 cDNA 的准确性，该测序结果显示了牛 E2 氨基末端和胰蛋白酶片段的肽序列。还以人 E2 cDNA 为探针，从 λZAP 载体的牛肝 cDNA 文库中分离出一个牛 E2 cDNA（0.7 kb）。用人 E2 cDNA 探针进行的 Northern 印迹分析表明，牛肝和人肾系膜细胞中的 E2 mRNA 大小分别为 3.3 kb 和 4.6 kb。源自人和牛 E2 cDNA 的一级结构显示，其前导序列包括起始甲硫氨酸以及由完整的含硫辛酰胺和二氢硫辛酰胺脱氢酶（E3）结合结构域及两个铰链区组成的同源成熟肽段。此外，人 E2 cDNA 包含部分 E2 核心内部序列、一个 3' - 非翻译区和一个 poly(A+)尾。将哺乳动物 E2 的推导氨基酸序列与大肠杆菌转乙酰基酶、转琥珀酰基酶以及牛肾转乙酰基酶的序列进行了比较。结果表明，硫辛酰胺连接位点侧翼序列和 E3 结合结构域具有高度保守性。文中提出了一些模型，以探讨 α - 酮酸脱氢酶复合体中含硫辛酰胺结构域和 E3 结合结构域保守的结构 - 功能关系的意义。
