Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Mar 1;346(Pt 2):413–421.

Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites.

L Agius 1, M Stubbs 1
PMCID: PMC1220868  PMID: 10677361

Abstract

Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.

Full Text

The Full Text of this article is available as a PDF (233.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agius L. Control of glucokinase translocation in rat hepatocytes by sorbitol and the cytosolic redox state. Biochem J. 1994 Feb 15;298(Pt 1):237–243. doi: 10.1042/bj2980237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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. doi: 10.1042/bj3250667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Agius L., Peak M., Alberti K. G. 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. doi: 10.1042/bj2660091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Agius L., Peak M., Newgard C. B., Gomez-Foix A. M., Guinovart J. J. 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. doi: 10.1074/jbc.271.48.30479. [DOI] [PubMed] [Google Scholar]
  6. Agius L., Peak M., Van Schaftingen E. The regulatory protein of glucokinase binds to the hepatocyte matrix, but, unlike glucokinase, does not translocate during substrate stimulation. Biochem J. 1995 Aug 1;309(Pt 3):711–713. doi: 10.1042/bj3090711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Agius L. The physiological role of glucokinase binding and translocation in hepatocytes. Adv Enzyme Regul. 1998;38:303–331. doi: 10.1016/s0065-2571(97)00001-0. [DOI] [PubMed] [Google Scholar]
  8. Aiston S., Trinh K. Y., Lange A. J., Newgard C. B., 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. doi: 10.1074/jbc.274.35.24559. [DOI] [PubMed] [Google Scholar]
  9. Becker T. C., Noel R. J., Johnson J. H., Lynch R. M., Hirose H., Tokuyama Y., Bell G. I., Newgard C. B. Differential effects of overexpressed glucokinase and hexokinase I in isolated islets. Evidence for functional segregation of the high and low Km enzymes. J Biol Chem. 1996 Jan 5;271(1):390–394. doi: 10.1074/jbc.271.1.390. [DOI] [PubMed] [Google Scholar]
  10. Brown K. S., Kalinowski S. S., Megill J. R., Durham S. K., Mookhtiar K. A. Glucokinase regulatory protein may interact with glucokinase in the hepatocyte nucleus. Diabetes. 1997 Feb;46(2):179–186. doi: 10.2337/diab.46.2.179. [DOI] [PubMed] [Google Scholar]
  11. Connolly B. A., Trayer I. P. Reaction of rat hepatic glucokinase with substrate-related and other alkylating agents. Eur J Biochem. 1979 Sep;99(2):299–308. doi: 10.1111/j.1432-1033.1979.tb13257.x. [DOI] [PubMed] [Google Scholar]
  12. Cárdenas M. L., Cornish-Bowden A., Ureta T. Evolution and regulatory role of the hexokinases. Biochim Biophys Acta. 1998 Mar 5;1401(3):242–264. doi: 10.1016/s0167-4889(97)00150-x. [DOI] [PubMed] [Google Scholar]
  13. Cárdenas M. L., Rabajille E., Niemeyer H. Suppression of kinetic cooperativity of hexokinase D (glucokinase) by competitive inhibitors. A slow transition model. Eur J Biochem. 1984 Nov 15;145(1):163–171. doi: 10.1111/j.1432-1033.1984.tb08536.x. [DOI] [PubMed] [Google Scholar]
  14. Holroyde M. J., Chesher J. M., Trayer I. P., Walker D. G. Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase. Biochem J. 1976 Feb 1;153(2):351–361. doi: 10.1042/bj1530351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iynedjian P. B. Mammalian glucokinase and its gene. Biochem J. 1993 Jul 1;293(Pt 1):1–13. doi: 10.1042/bj2930001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liang Y., Jetton T. L., Zimmerman E. C., Najafi H., Matschinsky F. M., Magnuson M. A. Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary. J Biol Chem. 1991 Apr 15;266(11):6999–7007. [PubMed] [Google Scholar]
  17. Machado de Domenech E. E., Sols A. Specificity of hexokinases towards some uncommon substrates and inhibitors. FEBS Lett. 1980 Sep 22;119(1):174–176. doi: 10.1016/0014-5793(80)81024-6. [DOI] [PubMed] [Google Scholar]
  18. Miwa I., Mitsuyama S., Toyoda Y., Murata T., Okuda J. High-yield purification of glucokinase from rat liver. Prep Biochem. 1990;20(2):163–178. doi: 10.1080/00327489008050187. [DOI] [PubMed] [Google Scholar]
  19. Niculescu L., Veiga-da-Cunha M., Van Schaftingen E. Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes. Biochem J. 1997 Jan 1;321(Pt 1):239–246. doi: 10.1042/bj3210239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Otieno S., Bhargava A. K., Barnard E. A., Ramel A. H. Essential thiols of yeast hexokinase: alkylation by a substrate-like reagent. Biochemistry. 1975 Jun 3;14(11):2403–2410. doi: 10.1021/bi00682a021. [DOI] [PubMed] [Google Scholar]
  21. Raushel F. M., Cleland W. W. Bovine liver fructokinase: purification and kinetic properties. Biochemistry. 1977 May 17;16(10):2169–2175. doi: 10.1021/bi00629a020. [DOI] [PubMed] [Google Scholar]
  22. SOLS A., CRANE R. K. Substrate specificity of brain hexokinase. J Biol Chem. 1954 Oct;210(2):581–595. [PubMed] [Google Scholar]
  23. Van Schaftingen E. A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate. Eur J Biochem. 1989 Jan 15;179(1):179–184. doi: 10.1111/j.1432-1033.1989.tb14538.x. [DOI] [PubMed] [Google Scholar]
  24. Van Schaftingen E. Short-term regulation of glucokinase. Diabetologia. 1994 Sep;37 (Suppl 2):S43–S47. doi: 10.1007/BF00400825. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Wilson J. E. An introduction to the isoenzymes of mammalian hexokinase types I-III. Biochem Soc Trans. 1997 Feb;25(1):103–107. doi: 10.1042/bst0250103. [DOI] [PubMed] [Google Scholar]
  27. Wilson J. E., Chung V. Rat brain hexokinase: further studies on the specificity of the hexose and hexose 6-phosphate binding sites. Arch Biochem Biophys. 1989 Mar;269(2):517–525. doi: 10.1016/0003-9861(89)90135-5. [DOI] [PubMed] [Google Scholar]
  28. Wilson J. E. Hexokinases. Rev Physiol Biochem Pharmacol. 1995;126:65–198. doi: 10.1007/BFb0049776. [DOI] [PubMed] [Google Scholar]
  29. van Schaftingen E., Veiga-da-Cunha M., Niculescu L. The regulatory protein of glucokinase. Biochem Soc Trans. 1997 Feb;25(1):136–140. doi: 10.1042/bst0250136. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES