Cornell N W, Janski A M, Rendon A
Fed Proc. 1985 May;44(8):2448-52.
Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase (EC 4.1.3.8) illustrate the information that uniquely can be obtained. Although the enzyme was previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were starved for 2 days, fed NIH stock diet ab libitum, or starved for 2 days and then refed a fat-free diet for 2 days, the noncytosolic activity was, respectively, 52, 21, or 24% of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 microM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis.
与传统的组织匀浆技术相比，用洋地黄皂苷对分离的肝细胞进行分级分离，能更快速地确定酶的区室化，在某些情况下，准确性也更高。以ATP柠檬酸裂解酶（EC 4.1.3.8）为例说明了这种独特的信息。尽管该酶以前被认为完全存在于细胞质中，但洋地黄皂苷分级分离显示，细胞内总ATP柠檬酸裂解酶的一部分与线粒体或其他结构结合，结合量随动物的营养状态而变化。在饥饿2天、随意喂食NIH标准饲料或饥饿2天然后再喂食2天无脂饲料的大鼠肝细胞中，非细胞质活性分别占细胞总裂解酶的52%、21%或24%。然而，由于饥饿/再喂养会极大地诱导脂肪生成酶，这种饮食状态下结合的裂解酶活性比饥饿或随意喂食的大鼠高10 - 12倍。柠檬酸裂解酶与亚细胞器的结合也受到辅酶A的影响。向洋地黄皂苷分级分离培养基中添加20微摩尔辅酶A会使所有裂解酶像细胞质酶一样从细胞中释放出来。相反，当用降血脂药物5 -（十四烷氧基）- 2 - 呋喃甲酸孵育肝细胞以降低细胞内游离辅酶A时，结合的裂解酶量会增加。这些结果表明非细胞质ATP柠檬酸裂解酶可能在脂肪生成中具有特殊作用。
