Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries.
Regulation of pig heart pyruvate dehydrogenase by phosphorylation. Studies on the subunit and phosphorylation stoicheiometries.
复制标题
通过磷酸化调节猪心丙酮酸脱氢酶。
DOI：
10.1042/bj1730659
复制
发表时间：
1978
期刊：
影响因子：
--
通讯作者：
P. J. Randle
中科院分区：
文献类型：
--
作者：
P. Sugden;P. J. Randle
1.用十二烷基硫酸钠/聚丙烯酰胺盘凝胶电泳法测定纯化的猪心丙酮酸脱氢酶复合体的亚基分子量为：丙酮酸脱羧酶，α-亚基40600，β-亚基35100，二氢硫辛基乙酰转移酶76100，二氢硫辛基脱氢酶58200。2.丙酮酸脱氢酶复合体被其完整的激酶灭活相当于掺入0.46nmolP/失活的复合体活性单位。3.磷酸盐进一步掺入到复合体中的极限为1.27nmolP/单位灭活的复合体(约.是失活所需时间的3倍)。4.磷酸只掺入脱羧酶的α-亚基中。5.用乙酰亚氨酸甲酯对丙酮酸脱氢酶[(32)P]磷酸复合体的氨基进行放射性酰胺化，然后用十二烷基硫酸钠/聚丙烯酰胺盘-凝胶电泳法分离α-亚基，估算了脱羧酶α-亚基与磷酸脱氢酶的摩尔比。该络合物的失活(0.46nmolP/单位失活的络合物)对应于一个磷酸基团与两个α链(即一个磷酸基团/α(2)β(2)四聚体)的摩尔比。完全磷酸化对应于每个α(2)β(2)四聚体的三个磷酸基团。6.用放射性胺化技术测定了络合物的亚基摩尔比。结果与4个α-亚基：4个β-亚基：2个二氢硫酰乙酰转移酶亚基：1个二氢硫酰脱氢酶亚基的摩尔比最接近。
1. The molecular weights of the subunits of purified pig heart pyruvate dehydrogenase complex were determined by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis and were: pyruvate decarboxylase, alpha-subunit 40600, beta-subunit 35100; dihydrolipoyl acetyltransferase 76100; dihydrolipoyl dehydrogenase 58200. 2. Inactivation of the pyruvate dehydrogenase complex by its integral kinase corresponded to the incorporation of 0.46nmol of P/unit of complex activity inactivated. 3. Further incorporation of phosphate into the complex occurred to a limit of 1.27nmol of P/unit of complex inactivated (approx. 3 times that required for inactivation). 4. Phosphate was incorporated only into the alpha-subunit of the decarboxylase. 5. The molar ratio of phosphate to alpha-subunits of the decarboxylase was estimated by radioamidination of amino groups of pyruvate dehydrogenase [(32)P]phosphate complex by using methyl [1-(14)C]acetimidate, followed by separation of alpha-subunits by sodium dodecyl sulphate/polyacrylamide-disc-gel electrophoresis. Inactivation of the complex (0.46nmol of P/unit of complex inactivated) corresponded to a molar ratio of one phosphate group per two alpha-chains (i.e. one phosphate group/alpha(2)beta(2) tetramer). Complete phosphorylation corresponded to three phosphate groups per alpha(2)beta(2) tetramer. 6. Subunit molar ratios in the complex were also estimated by the radioamidination technique. Results corresponded most closely to molar ratios of 4 alpha-subunits:4 beta-subunits:2 dihydrolipoyl acetyltransferase subunits:1 dihydrolipoyl dehydrogenase subunit.