Söling H D, Rescher C
Eur J Biochem. 1985 Feb 15;147(1):111-7. doi: 10.1111/j.1432-1033.1985.tb08726.x.
The discovery of a cold-labile cytosolic acetyl-CoA hydrolase of high activity in rat liver by Prass et al. [(1980) J. Biol. Chem. 255, 5215-5223] has questioned the importance of mitochondrial acetyl-CoA hydrolase for the formation of free acetate [Grigat et al. (1979) Biochem. J. 177, 71-79] under physiological conditions. Therefore this problem has been reevaluated by comparing various properties of the two enzymes. Cold-labile cytosolic acetyl-CoA hydrolase bands with an apparent Mr of 68000 during SDS/polyacrylamide gel electrophoresis, while the native enzyme elutes in two peaks with apparent Mr of 136000 and 245000 during gel chromatography in the presence of 2 mM ATP. The mitochondrial enzyme elutes under the same conditions with an apparent Mr of 157000. Under conditions where the cold-labile enzyme binds strongly to DEAE-Bio-Gel and ATP-agarose, the mitochondrial enzyme remains unbound. The cold-labile enzyme can be activated 14-fold by ATP, half-maximal activation occurring already at 40 microM ATP. AdoPP[NH]P, AdoPP[CH2]P and GTP have a similar though weaker effect. ADP as well as GDP can completely inhibit the cold-labile enzyme with 50% inhibition occurring for both nucleotides at about 1.45 microM. The binding of ATP and ADP is competitive. Acetyl phosphate and pyrophosphate have no effect on the activity of the cold-labile enzyme. The mitochondrial acetyl-CoA hydrolase is not affected by these nucleotides. CoASH is a strong product inhibitor (approximately equal to 80% inhibition at 40 microM CoASH) of the cold-labile enzyme, but only a weak inhibitor of the mitochondrial enzyme. Under in vivo conditions the activity of the cold-labile cytosolic acetyl-CoA hydrolase can be no more than 7% of the activity calculated for mitochondrial acetyl-CoA hydrolase under the same conditions. Accordingly the mitochondrial enzyme seems to be mainly responsible for the formation of free acetate by the intact liver, especially in view of the fact that the substrate specificity of the mitochondrial enzyme is much higher (activity ratios acetyl-CoA/butyryl-CoA 4.99 and 1.16 for the mitochondrial and the cold-labile enzyme respectively). Alloxan diabetes neither increased the activity of the cold-labile enzyme nor that of the mitochondrial enzyme. No experimental support has been found yet for the hypothesis that the acetyl-CoA hydrolase activity of the cold-labile enzyme represents the side-activity of an acetyl-transferase.
普拉斯等人[（1980年）《生物化学杂志》255卷，5215 - 5223页]在大鼠肝脏中发现了一种活性很高的冷不稳定胞质乙酰辅酶A水解酶，这对线粒体乙酰辅酶A水解酶在生理条件下对于游离乙酸形成的重要性提出了质疑[格里加特等人（1979年）《生物化学杂志》177卷，71 - 79页]。因此，通过比较这两种酶的各种特性对该问题进行了重新评估。冷不稳定胞质乙酰辅酶A水解酶在SDS/聚丙烯酰胺凝胶电泳中表观分子量为68000，而在2 mM ATP存在下进行凝胶过滤时，天然酶以表观分子量136000和245000的两个峰洗脱。线粒体酶在相同条件下以表观分子量157000洗脱。在冷不稳定酶与DEAE - Bio - Gel和ATP - 琼脂糖强烈结合的条件下，线粒体酶仍未结合。冷不稳定酶可被ATP激活14倍，在40 microM ATP时已出现半数最大激活。腺苷5′ - 磷酰基 - 5′ - 亚胺二磷酸（AdoPP[NH]P）、腺苷5′ - 磷酰基 - 5′ - 亚甲基二磷酸（AdoPP[CH2]P）和GTP有类似但较弱的作用。ADP以及GDP可完全抑制冷不稳定酶，两种核苷酸在约1.45 microM时均出现50%抑制。ATP和ADP的结合是竞争性的。乙酰磷酸和焦磷酸对冷不稳定酶的活性无影响。线粒体乙酰辅酶A水解酶不受这些核苷酸影响。辅酶A是冷不稳定酶的强产物抑制剂（在40 microM辅酶A时约80%抑制），但只是线粒体酶的弱抑制剂。在体内条件下，冷不稳定胞质乙酰辅酶A水解酶的活性不超过在相同条件下计算的线粒体乙酰辅酶A水解酶活性的7%。因此，线粒体酶似乎主要负责完整肝脏中游离乙酸的形成，特别是考虑到线粒体酶的底物特异性高得多（线粒体酶和冷不稳定酶的乙酰辅酶A/丁酰辅酶A活性比分别为4.99和1.16）这一事实。四氧嘧啶糖尿病既未增加冷不稳定酶的活性，也未增加线粒体酶的活性。尚未找到实验证据支持冷不稳定酶的乙酰辅酶A水解酶活性代表乙酰转移酶的副活性这一假说。
