Zhang B, Paxton R, Goodwin G W, Shimomura Y, Harris R A
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
Biochem J. 1987 Sep 15;246(3):625-31. doi: 10.1042/bj2460625.
A comparison was conducted of current methods for estimation of the activity states (proportion of enzyme in active, dephosphorylated, form) of hepatic branched-chain 2-oxo acid dehydrogenase. Practically all of the enzyme was active in freeze-clamped liver obtained from chow-fed and 48 h-starved rats, regardless of the presence of fluoride in the extraction and assay media to inhibit phosphatase activity. Likewise, the enzyme was almost completely active in mitochondria isolated by a conventional method from livers of chow-fed and starved rats. However, when fluoride and 4-methyl-2-oxopentanoate were included in the mitochondrial isolation medium the activity state was decreased to 73% and 47% in mitochondria isolated from chow-fed and starved rats respectively. Furthermore, branched-chain 2-oxo acid dehydrogenase became partially inactivated upon incubation of isolated mitochondria on ice in fluoride- and/or 4-methyl-2-oxopentanoate-supplemented media. The rate of inactivation was greater in mitochondria prepared from starved than from chow-fed rats, which correlated with the lower activity state found in mitochondria of starved rats isolated in the fluoride- and 4-methyl-2-oxopentanoate-supplemented media. Thus the activity state of branched-chain 2-oxo acid dehydrogenase is underestimated in mitochondria isolated in media supplemented with fluoride plus 4-methyl-2-oxopentanoate.
对目前用于估计肝脏支链2-氧代酸脱氢酶活性状态（活性、去磷酸化形式的酶比例）的方法进行了比较。实际上，在从正常饮食喂养和饥饿48小时的大鼠获得的冷冻钳夹肝脏中，几乎所有的酶都是有活性的，无论在提取和测定介质中是否存在氟化物以抑制磷酸酶活性。同样，通过常规方法从正常饮食喂养和饥饿大鼠的肝脏中分离出的线粒体中，该酶几乎完全有活性。然而，当线粒体分离介质中含有氟化物和4-甲基-2-氧代戊酸时，从正常饮食喂养和饥饿大鼠分离出的线粒体中的活性状态分别降至73%和47%。此外，在补充了氟化物和/或4-甲基-2-氧代戊酸的介质中，将分离出的线粒体在冰上孵育时，支链2-氧代酸脱氢酶会部分失活。饥饿大鼠制备的线粒体中的失活速率比正常饮食喂养大鼠的线粒体中的失活速率更大，这与在补充了氟化物和4-甲基-2-氧代戊酸的介质中分离出的饥饿大鼠线粒体中发现的较低活性状态相关。因此，在补充了氟化物加4-甲基-2-氧代戊酸的介质中分离出的线粒体中，支链2-氧代酸脱氢酶的活性状态被低估了。
