Complexes between hexokinase, outer membrane porin, and the adenylate translocator (ANT) were recently found to establish properties of the mitochondrial permeability transition pore in a reconstituted system. The complex was extracted by 0.5% Triton X-100 from rat brain membranes and separated by anion exchanger chromatography. The molecular weight was approximately 400kDa suggesting tetramers of hexokinase (monomer 100kDa). By the same method a porin, creatine kinase octamer, ANT complex was isolated and reconstituted in liposomes. Vesicles containing the reconstituted complexes both retained ATP that could be used by either kinase to phosphorylate external creatine or glucose. Atractyloside inhibited this activity indicating that the ANT was involved in this process and was functionally reconstituted [1]. Exclusively from the hexokinase complex containing liposome internal malate or ATP was released by addition of Ca2+ in a N-methylVal-4-cyclosporin sensitive way, suggesting that the hexokinase porin ANT complex might include the permeability transition pore (PTP). The Ca2+ dependent opening of the PTP-like structure was inhibited by ADP (apparent I50, 8μM) and ATP (apparent I50, 84μM). Also glucose inhibited the PTP-like activity, while glucose-6-phosphate abolished this effect. Although porin and ANT were functionally active in vesicles containing the creatine kinase octamer complex, Ca2+ did not induce a release of internal substrates. However, after dissociation of the creatine kinase octamer, the complex exhibited PTP-like properties and the vesicles liberated internal metabolites upon addition of Ca2+. The latter process was also inhibited by N-methylVal-4-cyclosporin. The activity of peptidyl-prolyl-cis-trans-isomerase (representing cyclophilin) was followed during complex isolation. Cyp D was co-purified with the hexokinase complex, while it was absent in the creatine kinase complex. The inhibitory effect of N-methylVal-4-cyclosporin on the creatine kinase complex may be explained by direct interaction with the creatine kinase dimer that appeared to support octamer formation.

Conference Article| February 01 1997 Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore G. Beutner; G. Beutner 1Faculty of Biology, University of Konstanz, D-78434 Konstanz, Germany Search for other works by this author on: This Site PubMed Google Scholar A. Rück; A. Rück 2Faculty of Biology, University of Konstanz, D-78434 Konstanz, Germany Search for other works by this author on: This Site PubMed Google Scholar B. Riede; B. Riede 3Faculty of Biology, University of Konstanz, D-78434 Konstanz, Germany Search for other works by this author on: This Site PubMed Google Scholar D. Brdiczka D. Brdiczka * 4Faculty of Biology, University of Konstanz, D-78434 Konstanz, Germany *To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Received: August 02 1996 Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1997 Biochemical Society1997 Biochem Soc Trans (1997) 25 (1): 151–157. https://doi.org/10.1042/bst0250151 Article history Received: August 02 1996 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation G. Beutner, A. Rück, B. Riede, D. Brdiczka; Complexes between hexokinase, mitochondrial porin and adenylate translocator in brain: regulation of hexokinase, oxidative phosphorylation and permeability transition pore. Biochem Soc Trans 1 February 1997; 25 (1): 151–157. doi: https://doi.org/10.1042/bst0250151 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: G6P, glucose 6-phosphate This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X‐100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel‐permeation chromatography, was approximately 400 kDa for both complexes. The M r suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCl. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca 2+ between 100 and 600 μM. The liberation of malate or ATP by Ca 2+ could be inhibited by N ‐methylVal‐4‐cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.