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. 1996 Feb 1;313(Pt 3):957–962. doi: 10.1042/bj3130957

Intracellular pH governs the subcellular distribution of hexokinase in a glioma cell line.

L Miccoli 1, S Oudard 1, F Sureau 1, F Poirson 1, B Dutrillaux 1, M F Poupon 1
PMCID: PMC1217004  PMID: 8611181

Abstract

Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pH1) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pH1 variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pH1 changes were verified microspectrofluorimetrically by using the pH1-sensitive probe SNARF-1-AM (seminaphtho-rhodafluor-1-acetoxymethyl ester). Imposition of an acidic pH1 for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pH1, was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pH1 and increased by basic pH1. Conversely, the glucose 6-phosphate level was increased by acidic pH1 and decreased by basic pH1. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pH1 was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pH1, and regulates hexokinase activity.

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Selected References

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  1. 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. doi: 10.1042/bj2960785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arora K. K., Pedersen P. L. Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP. J Biol Chem. 1988 Nov 25;263(33):17422–17428. [PubMed] [Google Scholar]
  3. Bachelard H. S., Lewis L. D., Pontén U., Siesjö B. K. Mechanisms activating glycolysis in the brain in arterial hypoxia. J Neurochem. 1974 Mar;22(3):395–401. doi: 10.1111/j.1471-4159.1974.tb07605.x. [DOI] [PubMed] [Google Scholar]
  4. Board M., Humm S., Newsholme E. A. Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells. Biochem J. 1990 Jan 15;265(2):503–509. doi: 10.1042/bj2650503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broniszewska-Ardelt B., Miller AT Jr+MILLER A. T., Jr Hypoxic changes in brain hexokinase distribution: phylogenetic and developmental considerations. Comp Biochem Physiol B. 1974 Sep 15;49(1B):151–156. doi: 10.1016/0305-0491(74)90234-x. [DOI] [PubMed] [Google Scholar]
  6. Bustamante E., Morris H. P., Pedersen P. L. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. J Biol Chem. 1981 Aug 25;256(16):8699–8704. [PubMed] [Google Scholar]
  7. Bustamante E., Pedersen P. L. High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3735–3739. doi: 10.1073/pnas.74.9.3735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cornish-Bowden A., Cárdenas M. L. Hexokinase and 'glucokinase' in liver metabolism. Trends Biochem Sci. 1991 Aug;16(8):281–282. doi: 10.1016/0968-0004(91)90115-c. [DOI] [PubMed] [Google Scholar]
  9. De Pinto V., Tommasino M., Benz R., Palmieri F. The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin. Biochim Biophys Acta. 1985 Mar 14;813(2):230–242. doi: 10.1016/0005-2736(85)90238-x. [DOI] [PubMed] [Google Scholar]
  10. Fagan J. B., Racker E. Determinants of glycolytic rate in normal and transformed chick embryo fibroblasts. Cancer Res. 1978 Mar;38(3):749–758. [PubMed] [Google Scholar]
  11. Felgner P. L., Messer J. L., Wilson J. E. Purification of a hexokinase-binding protein from the outer mitochondrial membrane. J Biol Chem. 1979 Jun 25;254(12):4946–4949. [PubMed] [Google Scholar]
  12. Felgner P. L., Wilson J. E. Effect of neutral salts on the interaction of rat brain hexokinase with the outer mitochondrial membrane. Arch Biochem Biophys. 1977 Jul;182(1):282–294. doi: 10.1016/0003-9861(77)90309-5. [DOI] [PubMed] [Google Scholar]
  13. Fidelman M. L., Seeholzer S. H., Walsh K. B., Moore R. D. Intracellular pH mediates action of insulin on glycolysis in frog skeletal muscle. Am J Physiol. 1982 Jan;242(1):C87–C93. doi: 10.1152/ajpcell.1982.242.1.C87. [DOI] [PubMed] [Google Scholar]
  14. Floridi A., Paggi M. G., Fanciulli M. Modulation of glycolysis in neuroepithelial tumors. J Neurosurg Sci. 1989 Jan-Mar;33(1):55–64. [PubMed] [Google Scholar]
  15. Galina A., Reis M., Albuquerque M. C., Puyou A. G., Puyou M. T., de Meis L. Different properties of the mitochondrial and cytosolic hexokinases in maize roots. Biochem J. 1995 Jul 1;309(Pt 1):105–112. doi: 10.1042/bj3090105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Griffiths J. R. Are cancer cells acidic? Br J Cancer. 1991 Sep;64(3):425–427. doi: 10.1038/bjc.1991.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gross J. L., Behrens D. L., Mullins D. E., Kornblith P. L., Dexter D. L. Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. Cancer Res. 1988 Jan 15;48(2):291–296. [PubMed] [Google Scholar]
  18. Gumaa K. A., McLean P. The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells. Biochem J. 1969 Dec;115(5):1009–1029. doi: 10.1042/bj1151009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Halperin M. L., Connors H. P., Relman A. S., Karnovsky M. L. Factors that control the effect of pH on glycolysis in leukocytes. J Biol Chem. 1969 Jan 25;244(2):384–390. [PubMed] [Google Scholar]
  20. Hochachka P. W., Mommsen T. P. Protons and anaerobiosis. Science. 1983 Mar 25;219(4591):1391–1397. doi: 10.1126/science.6298937. [DOI] [PubMed] [Google Scholar]
  21. Ion transport in liver mitochondria. Energy barrier and stoicheometry of aerobic K+ translocation. Eur J Biochem. 1969 Jan;7(3):418–426. doi: 10.1111/j.1432-1033.1969.tb19626.x. [DOI] [PubMed] [Google Scholar]
  22. JOHNSON M. K. The intracellular distribution of glycolytic and other enzymes in rat-brain homogenates and mitochondrial preparations. Biochem J. 1960 Dec;77:610–618. doi: 10.1042/bj0770610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaplan O., Navon G., Lyon R. C., Faustino P. J., Straka E. J., Cohen J. S. Effects of 2-deoxyglucose on drug-sensitive and drug-resistant human breast cancer cells: toxicity and magnetic resonance spectroscopy studies of metabolism. Cancer Res. 1990 Feb 1;50(3):544–551. [PubMed] [Google Scholar]
  24. Katzen H. M., Schimke R. T. Multiple forms of hexokinase in the rat: tissue distribution, age dependency, and properties. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1218–1225. doi: 10.1073/pnas.54.4.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Knull H. R., Taylor W. F., Wells W. W. Effects of energy metabolism on in vivo distribution of hexokinase in brain. J Biol Chem. 1973 Aug 10;248(15):5414–5417. [PubMed] [Google Scholar]
  26. Kyriazi H. T., Basford R. E. An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer mitochondrial membrane. Arch Biochem Biophys. 1986 Jul;248(1):253–271. doi: 10.1016/0003-9861(86)90423-6. [DOI] [PubMed] [Google Scholar]
  27. LOWRY O. H., PASSONNEAU J. V., HASSELBERGER F. X., SCHULZ D. W. EFFECT OF ISCHEMIA ON KNOWN SUBSTRATES AND COFACTORS OF THE GLYCOLYTIC PATHWAY IN BRAIN. J Biol Chem. 1964 Jan;239:18–30. [PubMed] [Google Scholar]
  28. Land J. M., Booth R. F., Berger R., Clark J. B. Development of mitochondrial energy metabolism in rat brain. Biochem J. 1977 May 15;164(2):339–348. doi: 10.1042/bj1640339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lindén M., Gellerfors P., Nelson B. D. Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical. FEBS Lett. 1982 May 17;141(2):189–192. doi: 10.1016/0014-5793(82)80044-6. [DOI] [PubMed] [Google Scholar]
  30. Lundin A., Hasenson M., Persson J., Pousette A. Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol. 1986;133:27–42. doi: 10.1016/0076-6879(86)33053-2. [DOI] [PubMed] [Google Scholar]
  31. Madshus I. H. Regulation of intracellular pH in eukaryotic cells. Biochem J. 1988 Feb 15;250(1):1–8. doi: 10.1042/bj2500001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Middleton R. J. Hexokinases and glucokinases. Biochem Soc Trans. 1990 Apr;18(2):180–183. doi: 10.1042/bst0180180. [DOI] [PubMed] [Google Scholar]
  33. Morgan-Hughes J. A., Darveniza P., Kahn S. N., Landon D. N., Sherratt R. M., Land J. M., Clark J. B. A mitochondrial myopathy characterized by a deficiency in reducible cytochrome b. Brain. 1977 Dec;100(4):617–640. doi: 10.1093/brain/100.4.617. [DOI] [PubMed] [Google Scholar]
  34. Oudard S., Poirson F., Miccoli L., Bourgeois Y., Vassault A., Poisson M., Magdelénat H., Dutrillaux B., Poupon M. F. Mitochondria-bound hexokinase as target for therapy of malignant gliomas. Int J Cancer. 1995 Jul 17;62(2):216–222. doi: 10.1002/ijc.2910620218. [DOI] [PubMed] [Google Scholar]
  35. Parry D. M., Pedersen P. L. Intracellular localization and properties of particulate hexokinase in the Novikoff ascites tumor. Evidence for an outer mitochondrial membrane location. J Biol Chem. 1983 Sep 25;258(18):10904–10912. [PubMed] [Google Scholar]
  36. Pedersen P. L. Tumor mitochondria and the bioenergetics of cancer cells. Prog Exp Tumor Res. 1978;22:190–274. doi: 10.1159/000401202. [DOI] [PubMed] [Google Scholar]
  37. Pouysségur J., Sardet C., Franchi A., L'Allemain G., Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4833–4837. doi: 10.1073/pnas.81.15.4833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reers M., Smith T. W., Chen L. B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry. 1991 May 7;30(18):4480–4486. doi: 10.1021/bi00232a015. [DOI] [PubMed] [Google Scholar]
  39. Rose I. A., Warms J. V. Mitochondrial hexokinase. Release, rebinding, and location. J Biol Chem. 1967 Apr 10;242(7):1635–1645. [PubMed] [Google Scholar]
  40. Seglen P. O. The effect of perfusate pH on respiration and glycolysis in the isolated rat liver perfused with an erythrocyte- and protein-free medium. Biochim Biophys Acta. 1972 May 16;264(3):398–410. doi: 10.1016/0304-4165(72)90002-5. [DOI] [PubMed] [Google Scholar]
  41. Seksek O., Henry-Toulmé N., Sureau F., Bolard J. SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment. Anal Biochem. 1991 Feb 15;193(1):49–54. doi: 10.1016/0003-2697(91)90042-r. [DOI] [PubMed] [Google Scholar]
  42. Smiley S. T., Reers M., Mottola-Hartshorn C., Lin M., Chen A., Smith T. W., Steele G. D., Jr, Chen L. B. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3671–3675. doi: 10.1073/pnas.88.9.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sprengers E. D., Koenderman A. H., Staal G. E. Mitochondrial and cytosolic hexokinase from rat brain: one and the same enzyme? Biochim Biophys Acta. 1983 Jan 4;755(1):112–118. doi: 10.1016/0304-4165(83)90280-5. [DOI] [PubMed] [Google Scholar]
  44. Sureau F., Moreau F., Millot J. M., Manfait M., Allard B., Aubard J., Schwaller M. A. Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells. Biophys J. 1993 Nov;65(5):1767–1774. doi: 10.1016/S0006-3495(93)81273-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tannock I. F., Rotin D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res. 1989 Aug 15;49(16):4373–4384. [PubMed] [Google Scholar]
  46. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  47. Thompson M. F., Bachelard H. S. Differences in catalytic properties between cerebral cytoplasmic and mitochondrial hexokinases. Biochem J. 1977 Mar 1;161(3):593–598. doi: 10.1042/bj1610593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Trivedi B., Danforth W. H. Effect of pH on the kinetics of frog muscle phosphofructokinase. J Biol Chem. 1966 Sep 10;241(17):4110–4112. [PubMed] [Google Scholar]
  49. Vandercammen A., Van Schaftingen E. Competitive inhibition of liver glucokinase by its regulatory protein. Eur J Biochem. 1991 Sep 1;200(2):545–551. doi: 10.1111/j.1432-1033.1991.tb16217.x. [DOI] [PubMed] [Google Scholar]
  50. Vaupel P., Kallinowski F., Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989 Dec 1;49(23):6449–6465. [PubMed] [Google Scholar]
  51. Wilson J. E. Brain hexokinase. A proposed relation between soluble-particulate distribution and activity in vivo. J Biol Chem. 1968 Jul 10;243(13):3640–3647. [PubMed] [Google Scholar]
  52. Wilson J. E. Hexokinases. Rev Physiol Biochem Pharmacol. 1995;126:65–198. doi: 10.1007/BFb0049776. [DOI] [PubMed] [Google Scholar]
  53. Wilson J. E. Ligand-induced conformations of rat brain hexokinase: effects of glucose-6-phosphate and inorganic phosphate. Arch Biochem Biophys. 1973 Nov;159(1):543–549. doi: 10.1016/0003-9861(73)90486-4. [DOI] [PubMed] [Google Scholar]
  54. Wilson J. E., Smith A. D. Monoclonal antibodies against rat brain hexokinase. Utilization in epitope mapping studies and establishment of structure-function relationships. J Biol Chem. 1985 Oct 15;260(23):12838–12843. [PubMed] [Google Scholar]

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