Guzman M, Kolodziej M P, Caldwell A, Corstorphine C G, Zammit V A
Department of Biochemistry and Molecular Biology I, Complutense University, Madrid, Spain.
Biochem J. 1994 Jun 15;300 ( Pt 3)(Pt 3):693-9. doi: 10.1042/bj3000693.
The mechanism of activation of mitochondrial overt carnitine palmitoyltransferase (CPT I) by treatment of hepatocytes with okadaic acid (OA) was investigated. Activation was observed when cells were permeabilized with digitonin, but not when a total membrane fraction was obtained by sonication. Both cell disruption methods preserved the activation of phosphorylase observed in OA-treated hepatocytes. Activation of CPT I was also observed in crude homogenates of OA-treated hepatocytes, but it was lost upon subsequent isolation of mitochondria from such homogenates. In all experiments, any activation observed did not depend on the presence or absence of fluoride ions in the permeabilization/homogenization media. When hepatocytes were permeabilized in the absence of fluoride and further incubated with exogenous phosphatases 1 and 2A, the OA-induced activation of CPT was not reversed, whereas the activation of glycogen phosphorylase in the same cells was rapidly reversed. Treatment of hepatocytes with OA, followed by permeabilization and incubation before assay of CPT I, demonstrated that OA had no short-term effect on the sensitivity of CPT I to malonyl-CoA, although the difference in sensitivity between cells isolated from fed and starved rats was fully preserved. Incubation of isolated mitochondria or purified mitochondrial outer membranes with cyclic AMP-dependent or AMP-activated protein kinases, under phosphorylating conditions, did not affect the activity of CPT I or its sensitivity to malonyl-CoA inhibition. Under the same conditions, the use of [32P]ATP resulted in the labelling of several outer-membrane proteins but, unlike [3H]etomoxir-labelled CPT I, none of them was specifically removed from membrane extracts by a specific polyclonal antibody to the enzyme. We conclude that the increase in overt CPT activity observed in permeabilized hepatocytes is not due to direct phosphorylation of CPT I, but may involve interactions between the mitochondrial outer membrane and other membranous or soluble cytosolic components of the cell.
研究了用冈田酸（OA）处理肝细胞激活线粒体明显肉碱棕榈酰转移酶（CPT I）的机制。当用洋地黄皂苷使细胞通透时观察到了激活现象，但通过超声处理获得总膜部分时则未观察到激活。两种细胞破碎方法均保留了在OA处理的肝细胞中观察到的磷酸化酶的激活。在OA处理的肝细胞的粗匀浆中也观察到了CPT I的激活，但随后从此类匀浆中分离线粒体时激活消失。在所有实验中，观察到的任何激活均不依赖于通透/匀浆介质中氟离子的存在与否。当肝细胞在无氟的情况下通透并进一步与外源性磷酸酶1和2A孵育时，OA诱导的CPT激活未被逆转，而同一细胞中糖原磷酸化酶的激活则迅速被逆转。用OA处理肝细胞，随后通透并在测定CPT I之前孵育，结果表明OA对CPT I对丙二酰辅酶A的敏感性没有短期影响，尽管从喂食和饥饿大鼠分离的细胞之间的敏感性差异得到了充分保留。在磷酸化条件下，将分离的线粒体或纯化的线粒体外膜与环磷酸腺苷依赖性或AMP激活的蛋白激酶一起孵育，不影响CPT I的活性或其对丙二酰辅酶A抑制的敏感性。在相同条件下，使用[32P]ATP导致几种外膜蛋白被标记，但与[3H]依托莫昔芬标记的CPT I不同，它们中没有一种能被针对该酶的特异性多克隆抗体从膜提取物中特异性去除。我们得出结论，在通透的肝细胞中观察到的明显CPT活性增加不是由于CPT I的直接磷酸化，而是可能涉及线粒体外膜与细胞的其他膜性或可溶性胞质成分之间的相互作用。
