Sheu K F, Hu C W, Utter M F
J Clin Invest. 1981 May;67(5):1463-71. doi: 10.1172/jci110176.
Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37 degrees C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-(14)C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of (14)CO(2) production on the presence of thiamin-PP, coenzyme A (CoA), Mg(++), and NAD(+). Also, it has been shown that acetyl-CoA and (14)CO(2) are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg(++) and Ca(++), or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency. Application of the same procedures to fibroblasts obtained from 16 individuals who were believed to have normal PDC activities showed a range from about 2-2.5 nmol/min per mg protein for adults to 5-6 nmol/min per mg protein for cells from infants.
与其他动物细胞一样，人类皮肤成纤维细胞中的丙酮酸脱氢酶复合体（PDC）活性似乎受磷酸化-去磷酸化机制调控。在细胞破碎以测量PDC活性之前，用丙酮酸脱氢酶激酶抑制剂二氯乙酸（DCA）预处理细胞，可激活该酶。经此处理后，婴儿成纤维细胞在37℃下的活性达到每毫克蛋白质5 - 6 nmol/分钟。这些数值相较于未处理细胞观察到的数值，代表了约5 - 20倍的激活。基于[1-(14)C]丙酮酸脱羧的该测定法能有效测量PDC的整体反应，这体现在(14)CO(2)的产生依赖于硫胺素-PP、辅酶A（CoA）、Mg(++)和NAD(+)的存在。此外，已表明乙酰辅酶A和(14)CO(2)以1:1的比例形成。通过添加纯化的丙酮酸脱氢酶磷酸酶以及高浓度的Mg(++)和Ca(++)，或者在某些情况下，在细胞破碎后向细胞匀浆中单独添加金属离子，也能实现类似程度的PDC激活。这些结果有力地表明激活是由于去磷酸化。添加抑制去磷酸化的NaF会导致PDC活性几乎完全丧失。对源自据报道该酶缺乏的患者的两个细胞系进行了完全激活的PDC测定（布拉斯，J.P.，J.阿维根，和B.W.乌布伦多夫。1970.《临床研究杂志》49: 423 - 432；布拉斯，J.P.，J.D.舒曼，D.S.扬，和E.哈姆。1972.《临床研究杂志》51: 1545 - 1551）。即便用DCA激活后，这些患者的成纤维细胞每毫克蛋白质的活性值仅为0.1和0.3 nmol/分钟。对其中一位患者的家族研究表明，父母双方在完全激活的细胞中的活性约为正常值的一半，而其一位兄弟姐妹的细胞看起来正常。这些结果证明了PDC缺乏的遗传性质，且目前的测定法足以检测该缺乏的杂合携带者。对从16名被认为具有正常PDC活性的个体获取的成纤维细胞应用相同程序，结果显示，成年人的活性范围约为每毫克蛋白质2 - 2.5 nmol/分钟，婴儿细胞的活性为每毫克蛋白质5 - 6 nmol/分钟。
