Patston P A, Espinal J, Shaw J M, Randle P J
Biochem J. 1986 Apr 15;235(2):429-34. doi: 10.1042/bj2350429.
A rabbit polyclonal antibody to purified ox kidney branched-chain oxo acid dehydrogenase complex was shown by a variety of techniques to be an antibody to the E2 (acyltransferase) component. Rocket immunoelectrophoresis showed that the antibody does not discriminate between phosphorylated (inactive) or dephosphorylated (active) complex, and the same technique is used to assay total branched-chain complex (sum of active and inactive forms) in rat liver and heart mitochondrial extracts. The values obtained in normal rats fed on normal diet were comparable with those obtained by spectrophotometric assay of the holocomplex reaction after conversion of inactive complex into active complex. The values obtained in liver mitochondria from rats fed on 0%-casein diet or starved for 48 h were comparable with those in rats fed on normal diet, whereas earlier studies using spectrophotometric assay had shown substantial decreases in rats fed on 0%-casein diet or starved for 48 h. It has been shown that conversion of inactive complex into active complex requires prolonged incubation (120 min) in the presence of ketoleucine (4-methyl-2-oxopentanoate; to inhibit branched-chain oxo acid dehydrogenase kinase) to effect complete conversion in mitochondria from rats fed on 0%-casein diet, or starved for 48 h, or made diabetic with alloxan. By this technique, total activity of the complex in rat liver mitochondria was unaffected by diet or diabetes. The effects of diet and diabetes to decrease the activity of branched-chain complex in rat liver are therefore apparently mediated wholly through inactivation of the complex by phosphorylation.
通过多种技术表明，一种针对纯化的牛肾支链氧代酸脱氢酶复合物的兔多克隆抗体是针对E2（酰基转移酶）成分的抗体。火箭免疫电泳显示，该抗体不能区分磷酸化（无活性）或去磷酸化（有活性）的复合物，并且相同的技术用于测定大鼠肝脏和心脏线粒体提取物中的总支链复合物（活性和无活性形式的总和）。在正常饮食喂养的正常大鼠中获得的值与在将无活性复合物转化为有活性复合物后通过分光光度法测定全酶复合物反应获得的值相当。在喂食0%酪蛋白饮食或饥饿48小时的大鼠肝脏线粒体中获得的值与正常饮食喂养的大鼠相当，而早期使用分光光度法的研究表明，喂食0%酪蛋白饮食或饥饿48小时的大鼠中该值大幅下降。已经表明，将无活性复合物转化为有活性复合物需要在酮亮氨酸（4-甲基-2-氧代戊酸；抑制支链氧代酸脱氢酶激酶）存在下长时间孵育（120分钟），以实现喂食0%酪蛋白饮食、饥饿48小时或用四氧嘧啶诱导糖尿病的大鼠线粒体中的完全转化。通过这种技术，大鼠肝脏线粒体中复合物的总活性不受饮食或糖尿病的影响。因此，饮食和糖尿病对大鼠肝脏中支链复合物活性降低的影响显然完全是通过复合物磷酸化失活介导的。
