Matuda S, Shirahama T, Saheki T, Miura S, Mori M
Biochim Biophys Acta. 1983 Oct 13;741(1):86-93. doi: 10.1016/0167-4781(83)90013-1.
Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.
丙酮酸脱氢酶复合体从大鼠心脏中纯化得到。该复合体在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳上显示出四条多肽带，分别对应丙酮酸脱氢酶的硫辛酰胺乙酰转移酶（分子量68000）、硫辛酰胺脱氢酶（分子量56000）、α亚基（分子量41000）和β亚基（分子量35000）。大鼠心脏丙酮酸脱氢酶复合体被解离为三种组成酶，并制备了针对每种组成酶的抗体。抗丙酮酸脱氢酶抗体和抗硫辛酰胺乙酰转移酶抗体能有效沉淀丙酮酸脱氢酶复合体，但抗硫辛酰胺脱氢酶抗体能从复合体中释放硫辛酰胺脱氢酶，并有效沉淀硫辛酰胺脱氢酶。硫辛酰胺脱氢酶是在无细胞网织红细胞裂解物系统中用大鼠肝脏的总RNA合成的。其翻译产物被检测为一种推测的前体，比成熟亚基大3000Da。在用游离和膜结合多聚核糖体进行的无细胞翻译中，编码该酶前体的mRNA活性在游离多聚核糖体中比在膜结合多聚核糖体中高得多。
