大鼠体内的四氧嘧啶糖尿病或饥饿会抑制心脏线粒体中通过磷酸反应将无活性（磷酸化）丙酮酸脱氢酶复合体转化为活性复合体的过程。

Conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active complex by the phosphate reaction in heart mitochondria is inhibited by alloxan-diabetes or starvation in the rat.

作者信息

Hutson N J, Kerbey A L, Randle P J, Sugden P H

出版信息

Biochem J. 1978 Aug 1;173(2):669-680. doi: 10.1042/bj1730669.

DOI:10.1042/bj1730669

PMID:212016

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1185822/

Abstract

The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.

摘要

丙酮酸脱氢酶磷酸酶将无活性（磷酸化）的丙酮酸脱氢酶复合物转化为有活性（去磷酸化）复合物的过程，在从四氧嘧啶糖尿病大鼠或饥饿48小时的大鼠制备的心脏线粒体中、在乙酸灌注的大鼠心脏制备的线粒体中以及在与呼吸底物孵育6分钟（与1分钟相比）的正常大鼠心脏制备的线粒体中均受到抑制。2. 这一结论基于以下实验：在分离的完整线粒体中，丙酮酸脱氢酶激酶反应通过丙酮酸或ATP耗竭（使用寡霉素和羰基氰化物间氯苯腙）而受到抑制；以及在实验中，在线粒体提取物中测量磷酸酶将无活性复合物转化为有活性复合物的速率。在Ca2+和Mg2+（磷酸酶的激活剂）浓度恒定的情况下，观察到磷酸酶反应受到抑制。当使用过量的外源性猪心脏丙酮酸脱氢酶磷酸作为底物时，这些线粒体提取物中的磷酸酶反应未受到抑制。得出的结论是，这种抑制是由于与底物（丙酮酸脱氢酶磷酸复合物）相关的某些因素，而不是对磷酸酶本身的抑制。3. 通过从对照大鼠和糖尿病大鼠的心脏以及从与呼吸底物孵育1分钟（对照）或6分钟的心脏线粒体中分离不含磷酸酶的丙酮酸脱氢酶磷酸复合物，验证了这一结论。然后用牛心脏或大鼠心脏磷酸酶制剂测量无活性复合物的再激活速率。来自糖尿病大鼠心脏或与呼吸底物孵育6分钟的心脏线粒体的无活性复合物的再激活速率（相对于对照）较低。4. 在大鼠心脏线粒体中，32Pi掺入无活性复合物需要6分钟才能完成。掺入程度与大鼠心脏丙酮酸脱氢酶复合物中三个或四个磷酸化位点一致。5. 有人提出，除失活位点外的其他位点的磷酸化可能会抑制从四氧嘧啶糖尿病大鼠或饥饿48小时的大鼠的心脏线粒体中或与呼吸底物孵育6分钟的线粒体中，磷酸酶将无活性复合物转化为有活性复合物的过程。