Schaffer W T, Olson M S
Biochem J. 1980 Nov 15;192(2):741-51. doi: 10.1042/bj1920741.
Studies were performed to elucidate factors involved in the regulation of pyruvate dehydrogenase activity in rat brain synaptosomes during membrane depolarization. Addition of 24 mM-KCl to synaptosomes resulted in increases in rates of O2 consumption (90%) and [1-(14)C]pyruvate decarboxylation (85%) and in the active/total ratio of extractable pyruvate dehydrogenase (90--100%) within 10 s. Neither pyruvate (10 mM) nor dichloroacetate (10 mM) affected the activation state of the enzyme complex. Also, the activation state of pyruvate dehydrogenase was unaffected by addition of 1 mM-octanoate, L-(--)-carnitine, 3-hydroxybutyrate, glutamate, citrate, lactate, L-malate, acetate, acetaldehyde or ethanol. Removal of Ca2+ by using EGTA lowered the active/total ratio to about 70%, although the rate of O2 consumption and pyruvate decarboxylation was unaffected. Rates of pyruvate decarboxylation in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence and absence of NaF and EGTA demonstrated a linear correlation with changes in the activity of the enzyme complex. This observation indicated that a change in the activation state of pyruvate dehydrogenase from 90 to 100% active could result in a 27% increase in the rate of pyruvate decarboxylation. It is suggested that the pyruvate dehydrogenase complex is an important site for the regulation of substrate utilization in rat brain synaptosomes. Further, the phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.
开展了多项研究以阐明大鼠脑突触体在膜去极化过程中丙酮酸脱氢酶活性调节所涉及的因素。向突触体中添加24 mM - KCl会导致在10秒内氧气消耗速率增加90%、[1-(14)C]丙酮酸脱羧速率增加85%以及可提取的丙酮酸脱氢酶的活性/总活性比值增加90 - 100%。10 mM的丙酮酸或10 mM的二氯乙酸均不影响酶复合物的激活状态。此外，添加1 mM的辛酸、L-( - )-肉碱、3-羟基丁酸、谷氨酸、柠檬酸、乳酸、L-苹果酸、乙酸、乙醛或乙醇对丙酮酸脱氢酶的激活状态也无影响。使用乙二醇双(2-氨基乙醚)四乙酸(EGTA)去除Ca2 +会使活性/总活性比值降至约70%，尽管氧气消耗速率和丙酮酸脱羧速率未受影响。在存在和不存在氟化钠(NaF)及EGTA的情况下，羰基氰对三氟甲氧基苯腙存在时的丙酮酸脱羧速率与酶复合物活性变化呈线性相关。这一观察结果表明，丙酮酸脱氢酶的激活状态从90%变为100%活跃可导致丙酮酸脱羧速率增加27%。有人提出丙酮酸脱氢酶复合物是大鼠脑突触体中底物利用调节的一个重要位点。此外，磷酸化/去磷酸化系统以及对酶复合物的直接反馈抑制作用在使丙酮酸脱羧迅速适应线粒体能量产生需求的变化方面均发挥着重要作用。
