title: Activation of low Km hexokinases in purified hepatocytes by binding to mitochondria.
abstract: 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.
authors.affiliation: Faculty of Biology, University of Konstanz, F.R.G.
authors.forename: V
authors.initials: V
authors.lastname: Adams
authors.forename: W
authors.initials: W
authors.lastname: Bosch
authors.forename: T
authors.initials: T
authors.lastname: Hämmerle
authors.forename: D
authors.initials: D
authors.lastname: Brdiczka
mesh_terms: D000818:Animals; D002469:Cell Separation; D004789:Enzyme Activation; D005260:Female; D006593:Hexokinase / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats
chemical_list: D007527:Isoenzymes; D006593:Hexokinase