[32P]磷酸盐掺入大鼠心脏线粒体丙酮酸脱氢酶复合物的研究。

Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria.

作者信息

Sale G J, Randle P J

出版信息

Biochem J. 1980 May 15;188(2):409-21. doi: 10.1042/bj1880409.

DOI:10.1042/bj1880409

PMID:7396870

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1161884/

Abstract

Evidence is given for three sites of phosphorylation in the alpha-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. 2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1, 2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. 3. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations ranging from 0.15 to 0.4 mM. The results showed that the concentration of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 greater than site 2 greater than site 1. 4. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporation of 32Pi into site 3, but it had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. It is concluded that site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. 5. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.

摘要

已证实纯化的大鼠心脏丙酮酸脱氢酶复合体脱羧酶组分的α链中有三个磷酸化位点，这与猪和牛的复合体中已确定的位点类似。大鼠心脏复合体的失活与位点1的磷酸化相关。磷酸化的相对初始速率为位点1＞位点2＞位点3。2. 描述了测量32Pi掺入大鼠心脏线粒体中氧化2-氧代戊二酸+L-苹果酸的复合体（总量，位点1、2和3）的方法。在正常或糖尿病大鼠的线粒体中，复合体的失活与位点1的磷酸化呈线性相关。磷酸化的相对初始速率为位点1＞位点2＞位点3。糖尿病大鼠线粒体中位点2和位点3的磷酸化速率可能比正常大鼠线粒体中更接近位点1的速率。3. 通过用0.15至0.4 mM浓度的丙酮酸抑制激酶反应，改变正常大鼠线粒体中无活性（磷酸化）复合体的浓度。结果表明，无活性复合体的浓度与32Pi掺入位点1呈线性相关。在此浓度范围内，丙酮酸对32Pi掺入的抑制作用为位点3＞位点2＞位点。4. 对于糖尿病大鼠的线粒体，丙酮酸（0.15 - 0.4 mM）抑制32Pi掺入位点3，但对无活性复合体的浓度或32Pi掺入位点1或位点2没有影响。得出的结论是，大鼠心脏线粒体中复合体的失活不需要位点3的磷酸化。5. 有证据表明，位点2和位点3的磷酸化可能会抑制大鼠心脏线粒体中复合体通过去磷酸化的再激活。