大鼠肝脏支链α-酮酸脱氢酶的生理性共价调节

Physiological covalent regulation of rat liver branched-chain alpha-ketoacid dehydrogenase.

作者信息

Harris R A, Powell S M, Paxton R, Gillim S E, Nagae H

出版信息

Arch Biochem Biophys. 1985 Dec;243(2):542-55. doi: 10.1016/0003-9861(85)90531-4.

Abstract

A radiochemical assay was developed for measuring branched-chain alpha-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain alpha-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain alpha-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain alpha-ketoacids, established inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain alpha-ketoacid dehydrogenase kinase by branched-chain alpha-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain alpha-ketoacid dehydrogenase activity state.

摘要

我们开发了一种放射化学分析法，用于测定冷冻钳夹的大鼠肝脏经 Triton X - 100 提取后的支链α-酮酸脱氢酶活性。通过测量用广谱特异性磷蛋白磷酸酶激活复合物前后的酶活性，来确定活性（去磷酸化）酶的比例。正常雄性 Wistar 大鼠肝脏中的支链α-酮酸脱氢酶活性有 97%处于活性状态，但在低蛋白（8%）饮食 2 天后降至 33%活性，在相同饮食 4 天后降至 13%活性。限制瘦型和肥胖型雌性 Zucker 大鼠的蛋白质摄入量也会导致肝脏支链α-酮酸脱氢酶复合物失活。在蛋白质含量为 30%或 50%的饮食中维持 14 天的大鼠，基本上所有的酶都处于活性状态。以商业普通饲料（最低 23%蛋白质）喂养的大鼠也是如此。然而，将大鼠维持在 20%、8%和 0%蛋白质饮食中，会使酶的活性形式占总酶的百分比分别降至 58%、10%和 7%。以普通饲料喂养的大鼠禁食 48 小时对肝脏支链α-酮酸脱氢酶的活性状态没有影响，即与以普通饲料喂养的大鼠的 97%相比，93%的酶仍处于活性状态。然而，以 8%蛋白质饮食喂养的大鼠，其肝脏酶在饥饿前为 10%活性，饥饿 2 天后为 83%活性。因此，饮食蛋白质缺乏会导致肝脏支链α-酮酸脱氢酶复合物失活，推测这是由于肝脏中支链α-酮酸水平较低所致，支链α-酮酸是支链α-酮酸脱氢酶激酶的既定抑制剂。对于喂食低蛋白饮食随后又饥饿的大鼠，内源性蛋白质水解产生的支链α-酮酸对支链α-酮酸脱氢酶激酶的抑制作用很可能促进了更高的支链α-酮酸脱氢酶活性状态。