Adams V, Bosch W, Hämmerle T, Brdiczka D
Faculty of Biology, University of Konstanz, F.R.G.
Biochim Biophys Acta. 1988 Feb 11;932(2):195-205. doi: 10.1016/0005-2728(88)90156-9.
Hepatocytes were purified on a Percoll gradient. The cell membrane of these hepatocytes was disrupted by digitonin in the presence of albumin, glucose and physiological concentrations of monovalent and divalent cations. This treatment led to a separation between free and loosely structure-bound cytosolic enzymes which is not achieved by conventional subfractionation techniques. According to kinetic and immunological analyses, the free extractable cytosolic fraction contained high Km, hexokinase (glucokinase) and less than 10% of low Km hexokinases, while the hexokinase activity bound to the cell structures represented exclusively low Km isozymes. The total activity of the bound hexokinases was comparable to that observed in the supernate (approx. 1.0 U per g fresh weight). This activity decreased more than 10-fold upon desorption at higher digitonin concentrations. Such activation by binding, as well as inactivation by desorption, could also be demonstrated in intact hepatocytes correlated to different metabolic states, and also in vitro with isolated mitochondria and purified isozyme I. The binding of low Km hexokinases in hepatocytes was restricted to the mitochondrial fraction and there it was observed in the contact sites between the two mitochondrial boundary membranes. In view of these findings it appears that the binding-dissociation equilibrium of low Km hexokinases plays an important role in metabolic regulation of glucose uptake and glycogen synthesis in the liver and presumably in muscle.
肝细胞在Percoll梯度上进行纯化。在白蛋白、葡萄糖以及生理浓度的单价和二价阳离子存在的情况下，用洋地黄皂苷破坏这些肝细胞的细胞膜。这种处理导致游离的和结构松散结合的胞质酶之间的分离，这是传统亚分级分离技术所无法实现的。根据动力学和免疫学分析，游离的可提取胞质组分含有高Km的己糖激酶（葡萄糖激酶），且低Km己糖激酶含量低于10%，而与细胞结构结合的己糖激酶活性仅代表低Km同工酶。结合的己糖激酶的总活性与上清液中观察到的活性相当（约每克鲜重1.0 U）。在较高浓度的洋地黄皂苷解吸时，这种活性下降超过10倍。这种通过结合的激活以及通过解吸的失活，在与不同代谢状态相关的完整肝细胞中以及在体外与分离的线粒体和纯化的同工酶I中也得到了证实。肝细胞中低Km己糖激酶的结合仅限于线粒体部分，并且在两个线粒体边界膜之间的接触部位观察到这种结合。鉴于这些发现，低Km己糖激酶的结合-解离平衡似乎在肝脏以及可能在肌肉中葡萄糖摄取和糖原合成的代谢调节中起重要作用。
