Chiang P K, Sacktor B
J Biol Chem. 1975 May 10;250(9):3399-408.
The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..
在从兔心脏分离出的完整线粒体中,研究了通过失活和激活来控制丙酮酸脱氢酶活性的情况。通过将线粒体与ATP、寡霉素和NaF一起孵育,丙酮酸脱氢酶可以被完全失活。脱氢酶活性的这种丧失与[γ-32P]ATP中的32P掺入线粒体蛋白以及丙酮酸的线粒体氧化减少相关。ATP可以外源供应,在氧化磷酸化过程中由内源性ADP产生,或者在羰基氰化物对三氟甲氧基苯基腙解偶联的线粒体中由外源ADP形成。对于偶联的线粒体,使脱氢酶半失活所需添加的ATP浓度为0.24 mM。对于解偶联的线粒体,表观Km降至60 μM ATP。外源ATP使丙酮酸脱氢酶失活对苍术苷敏感,这表明丙酮酸脱氢酶激酶在苍术苷敏感屏障内部起作用。二价阳离子载体A23187增强了脱氢酶活性的丧失。丙酮酸脱氢酶活性还受到丙酮酸、无机磷酸盐和ADP的额外调节。在存在鱼藤酮的情况下,丙酮酸强烈抑制失活。这表明丙酮酸促进其自身的氧化,并且底物增加丙酮酸脱氢酶活性可能对通过三羧酸循环利用丙酮酸提供调节作用。无机磷酸盐保护脱氢酶不被ATP失活。与ATP一起添加到孵育混合物中的ADP抑制丙酮酸脱氢酶的失活。这种保护可能源于对丙酮酸脱氢酶激酶的直接作用,因为ADP与ATP竞争,以及间接作用,即ADP与ATP竞争转位酶。有人认为线粒体内的[ATP]:[ADP]比值直接影响激酶活性,而胞质中的[ATP]:[ADP]比值则间接起作用。Mg2+可能通过加速酶的去磷酸化速率来提高失活的丙酮酸脱氢酶的再激活速率。添加0.5 mM Mg2+可获得最大激活。