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Biochem J. Dec 15, 2000; 352(Pt 3): 899–905.
PMCID: PMC1221532

Use of alpha-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes.

Abstract

We investigated whether hepatocytes permeabilized with alpha-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2, 6-bisphosphate [Fru(2,6)P(2)], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25 mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P(2), rates of detritiation of [2-(3)H]glucose and [3-(3)H]glucose were similar. Exogenous Glc6P (1 mM) and to a lesser extent fructose 6-phosphate, but not Fru(1, 6)P(2), decreased the rate of detritiation of [3-(3)H]glucose. During incubation with 25 mM glucose and Glc6P (0.2-1 mM), with either [3-(3)H]glucose or [3-(3)H]Glc6P as labelled substrate, there was dilution of metabolism of [3-(3)H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P(2). They provide an alternative explanation for the concept that there is more than one Glc6P pool.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Agius L, Peak M, Newgard CB, Gomez-Foix AM, Guinovart JJ. Evidence for a role of glucose-induced translocation of glucokinase in the control of hepatic glycogen synthesis. J Biol Chem. 1996 Nov 29;271(48):30479–30486. [PubMed]
  • Aiston S, Trinh KY, Lange AJ, Newgard CB, Agius L. Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase. J Biol Chem. 1999 Aug 27;274(35):24559–24566. [PubMed]
  • Van Schaftingen E, Detheux M, Veiga da Cunha M. Short-term control of glucokinase activity: role of a regulatory protein. FASEB J. 1994 Apr 1;8(6):414–419. [PubMed]
  • de la Iglesia N, Mukhtar M, Seoane J, Guinovart JJ, Agius L. The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte. J Biol Chem. 2000 Apr 7;275(14):10597–10603. [PubMed]
  • Seoane J, Gómez-Foix AM, O'Doherty RM, Gómez-Ara C, Newgard CB, Guinovart JJ. Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase. J Biol Chem. 1996 Sep 27;271(39):23756–23760. [PubMed]
  • Agius L. Involvement of glucokinase translocation in the mechanism by which resorcinol inhibits glycolysis in hepatocytes. Biochem J. 1997 Aug 1;325(Pt 3):667–673. [PMC free article] [PubMed]
  • Seoane J, Trinh K, O'Doherty RM, Gómez-Foix AM, Lange AJ, Newgard CB, Guinovart JJ. Metabolic impact of adenovirus-mediated overexpression of the glucose-6-phosphatase catalytic subunit in hepatocytes. J Biol Chem. 1997 Oct 24;272(43):26972–26977. [PubMed]
  • Villar-Palasí C, Guinovart JJ. The role of glucose 6-phosphate in the control of glycogen synthase. FASEB J. 1997 Jun;11(7):544–558. [PubMed]
  • Hers HG, Van Schaftingen E. Fructose 2,6-bisphosphate 2 years after its discovery. Biochem J. 1982 Jul 15;206(1):1–12. [PMC free article] [PubMed]
  • Hue L, Rider MH. Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem J. 1987 Jul 15;245(2):313–324. [PMC free article] [PubMed]
  • Srere PA. Complexes of sequential metabolic enzymes. Annu Rev Biochem. 1987;56:89–124. [PubMed]
  • Orosz F, Ovádi J. A simple approach to identify the mechanism of intermediate transfer: enzyme system related to triose phosphate metabolism. Biochim Biophys Acta. 1987 Sep 2;915(1):53–59. [PubMed]
  • Masters CJ. Interactions between soluble enzymes and subcellular structure. CRC Crit Rev Biochem. 1981;11(2):105–143. [PubMed]
  • Ovádi J. Physiological significance of metabolic channelling. J Theor Biol. 1991 Sep 7;152(1):1–22. [PubMed]
  • Hardin CD, Finder DR. Glycolytic flux in permeabilized freshly isolated vascular smooth muscle cells. Am J Physiol. 1998 Jan;274(1 Pt 1):C88–C96. [PubMed]
  • Clegg JS, Jackson SA. Glycolysis in permeabilized L-929 cells. Biochem J. 1988 Oct 1;255(1):335–344. [PMC free article] [PubMed]
  • Clegg JS, Jackson SA. Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells. Biochem Biophys Res Commun. 1989 May 15;160(3):1409–1414. [PubMed]
  • Clegg JS, Jackson SA. Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells. Arch Biochem Biophys. 1990 May 1;278(2):452–460. [PubMed]
  • Valeva A, Weisser A, Walker B, Kehoe M, Bayley H, Bhakdi S, Palmer M. Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin. EMBO J. 1996 Apr 15;15(8):1857–1864. [PMC free article] [PubMed]
  • Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science. 1996 Dec 13;274(5294):1859–1866. [PubMed]
  • Maechler P, Wollheim CB. Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis. Nature. 1999 Dec 9;402(6762):685–689. [PubMed]
  • Kadowaki M, Venerando R, Miotto G, Mortimore GE. De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs. J Biol Chem. 1994 Feb 4;269(5):3703–3710. [PubMed]
  • Stals HK, Mannaerts GP, Declercq PE. Factors influencing triacylglycerol synthesis in permeabilized rat hepatocytes. Biochem J. 1992 May 1;283(Pt 3):719–725. [PMC free article] [PubMed]
  • Cheung CW, Cohen NS, Raijman L. Channeling of urea cycle intermediates in situ in permeabilized hepatocytes. J Biol Chem. 1989 Mar 5;264(7):4038–4044. [PubMed]
  • Harshman S, Sugg N, Cassidy P. Preparation and purification of staphylococcal alpha toxin. Methods Enzymol. 1988;165:3–7. [PubMed]
  • Agius L, Peak M, Alberti KG. Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes. Biochem J. 1990 Feb 15;266(1):91–102. [PMC free article] [PubMed]
  • Agius L, Peak M. Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin. Biochem J. 1993 Dec 15;296(Pt 3):785–796. [PMC free article] [PubMed]
  • Clifton PM, Chang L, Mackinnon AM. Development of an automated Lowry protein assay for the Cobas-Bio centrifugal analyzer. Anal Biochem. 1988 Jul;172(1):165–168. [PubMed]
  • Hue L. The role of futile cycles in the regulation of carbohydrate metabolism in the liver. Adv Enzymol Relat Areas Mol Biol. 1981;52:247–331. [PubMed]
  • Cohen NS, Cheung CW, Raijman L. Channeling of extramitochondrial ornithine to matrix ornithine transcarbamylase. J Biol Chem. 1987 Jan 5;262(1):203–208. [PubMed]
  • Cohen NS, Cheung CW, Sijuwade E, Raijman L. Kinetic properties of carbamoyl-phosphate synthase (ammonia) and ornithine carbamoyltransferase in permeabilized mitochondria. Biochem J. 1992 Feb 15;282(Pt 1):173–180. [PMC free article] [PubMed]
  • Agius L. Substrate modulation of aldolase B binding in hepatocytes. Biochem J. 1996 Apr 15;315(Pt 2):651–658. [PMC free article] [PubMed]
  • Pagliaro L, Taylor DL. Aldolase exists in both the fluid and solid phases of cytoplasm. J Cell Biol. 1988 Sep;107(3):981–991. [PMC free article] [PubMed]
  • Brown KS, Kalinowski SS, Megill JR, Durham SK, Mookhtiar KA. Glucokinase regulatory protein may interact with glucokinase in the hepatocyte nucleus. Diabetes. 1997 Feb;46(2):179–186. [PubMed]
  • Mukhtar M, Stubbs M, Agius L. Evidence for glucose and sorbitol-induced nuclear export of glucokinase regulatory protein in hepatocytes. FEBS Lett. 1999 Dec 3;462(3):453–458. [PubMed]
  • Niemeyer H, Cerpa C, Rabajille E. Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver. Arch Biochem Biophys. 1987 Aug 15;257(1):17–26. [PubMed]
  • Niemeyer H, Rabajille E. Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts. Arch Biochem Biophys. 1988 Aug 15;265(1):91–93. [PubMed]
  • Sims EA, Landau BR. Insulin responsive and nonresponsive pools of glucose 6-phosphate in diaphragmatic muscle. Fed Proc. 1966 May-Jun;25(3):835–839. [PubMed]
  • Lynch RM, Paul RJ. Compartmentation of carbohydrate metabolism in vascular smooth muscle: evidence for at least two functionally independent pools of glucose 6-phosphate. Biochim Biophys Acta. 1986 Aug 1;887(3):315–318. [PubMed]
  • Christ B, Jungermann K. Sub-compartmentation of the 'cytosolic' glucose 6-phosphate pool in cultured rat hepatocytes. FEBS Lett. 1987 Sep 14;221(2):375–380. [PubMed]

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