Lynen A, Sedlaczek E, Wieland O H
Biochem J. 1978 Feb 1;169(2):321-28. doi: 10.1042/bj1690321.
An enzyme inactivating the pyruvate dehydrogenase complex (inactivase) was purified about 8000-fold from rat liver by differential centrifugation, acid extraction of a lysosomerich 25000 g pellet, acetone fractionation, and adsorption on calcium phosphate gel. By exclusion chromatography on Sephadex G-100 a molecular weight of 21 000 was estimated. The purified enzyme was most stable at pH 5.8 in potassium phosphate buffer, and at pH 4.5 in McIlvaine buffer. At high dilutions the enzyme was very labile and was remarkably stabilized by high salt concentrations. Enzyme activity is inhibited by native rat blood serum, iodoacetamide and leupeptin, but not by phenylmethanesulphonyl fluoride, suggesting that it belongs to the class of thiol proteinases. Among various enzymes tested, only 2-oxoglutarate dehydrogenase was attacked by the inactivase to a similar extent to the pyruvate dehydrogenase complex. Studies on the inactivation mechanism indicate that although the overall reaction is completely lost after treatment with inactivase, each individual step of the multienzyme complex retains full catalytic activity. As judged from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the transacetylase subunit appears to be degraded into several smaller fractions.
一种使丙酮酸脱氢酶复合体失活的酶（失活酶）通过差速离心、对富含溶酶体的25000g沉淀进行酸提取、丙酮分级分离以及吸附于磷酸钙凝胶，从大鼠肝脏中纯化了约8000倍。通过Sephadex G - 100凝胶过滤色谱法估计其分子量为21000。纯化后的酶在磷酸钾缓冲液中pH 5.8时最稳定，在McIlvaine缓冲液中pH 4.5时最稳定。在高稀释度下，该酶非常不稳定，而高盐浓度能显著使其稳定。酶活性受到天然大鼠血清、碘乙酰胺和亮抑酶肽的抑制，但不受苯甲基磺酰氟的抑制，这表明它属于巯基蛋白酶类。在测试的各种酶中，只有2 - 氧代戊二酸脱氢酶受到失活酶的攻击程度与丙酮酸脱氢酶复合体相似。对失活机制的研究表明，尽管用失活酶处理后整体反应完全丧失，但多酶复合体的每个单独步骤仍保留完全的催化活性。从十二烷基硫酸钠/聚丙烯酰胺凝胶电泳判断，转乙酰酶亚基似乎被降解成几个较小的片段。
