Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Apr 15;315(Pt 2):651–658. doi: 10.1042/bj3150651

Substrate modulation of aldolase B binding in hepatocytes.

L Agius 1
PMCID: PMC1217246  PMID: 8615843

Abstract

The binding properties of hepatic aldolase (B) were determined in digitonin-permeabilized rat hepatocytes after the cells had been preincubated with either glycolytic or gluconeogenic substrates. In hepatocytes that had been preincubated in medium containing 5 mM glucose as sole carbohydrate substrate, binding of aldolase to the hepatocyte matrix was maximal at low KCl concentrations (20 mM) or bivalent cation concentrations (1 mM Mg2+) and half-maximal dissociation occurred at 50 mM KCl. Preincubation of hepatocytes (for 10-30 min) with glucose or mannose (10-40 mM), fructose, sorbitol, dihydroxyacetone or glycerol (1-10 mM), caused a leftward shift of the salt dissociation curve (maximum binding at 10 mM KCl; half-maximum dissociation at 35 mM KCl) but did not affect the proportion of bound enzyme at low or high KCl concentrations. Galactose and 2-deoxyglucose had no effect on aldolase binding. Inhibitors of glucokinase (mannoheptulose and glucosamine) suppressed the effects of glucose but not the effects of sorbitol, glycerol or dihydroxyacetone. Glucagon suppressed the effects of glucose, fructose and dihydroxyacetone but not glycerol. Poly(ethylene glycol) (PEG) (2-10%), added to the permeabilization medium, increased aldolase binding and caused a rightward shift in the salt dissociation curve. In the presence of PEG (6-8%), the effects of substrates on aldolase dissociation were shifted to higher salt concentrations (50-100 mM versus 35 mM KCl). The effects of substrates (added to the intact cell) on aldolase binding to the permeabilized cell could be mimicked by addition of the phosphorylated derivatives of these substrates to the permeabilized cell. Of the intermediates tested dihydroxyacetone phosphate and fructose 1,6-bisphosphate were the most effective at dissociating aldolase (A50 values of 20 microM and 40 microM respectively). Other effective intermediates in order of decreasing potency were fructose 1-phosphate, glycerol 3-phosphate, glucose 1,6-bisphosphate/fructose 2,6-bisphosphate. These results show that aldolase B binds to the hepatocyte matrix by a salt-dependent mechanism that is influenced by macromolecular crowding and metabolic intermediates. Maximum binding occurs when hepatocytes are incubated in the absence of glycolytic and gluconeogenic substrates and minimum binding occurs in the presence of substrates that are precursors of either fructose 1,6-bisphosphate or triose phosphates. Since the bound form of aldolase represents a kinetically less active state it is proposed that aldolase binding and dissociation may be a mechanism for buffering the concentrations of metabolic intermediates.

Full Text

The Full Text of this article is available as a PDF (605.1 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. Hexokinase and glucokinase binding in permeabilized guinea-pig hepatocytes. Biochem J. 1994 Nov 1;303(Pt 3):841–846. doi: 10.1042/bj3030841. [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. Aridor-Piterman O., Lavie Y., Liscovitch M. Bimodal distribution of phosphatidic acid phosphohydrolase in NG108-15 cells. Modulation by the amphiphilic lipids oleic acid and sphingosine. Eur J Biochem. 1992 Mar 1;204(2):561–568. doi: 10.1111/j.1432-1033.1992.tb16668.x. [DOI] [PubMed] [Google Scholar]
  6. Arnold H., Pette D. Binding of aldolase and triosephosphate dehydrogenase to F-actin and modification of catalytic properties of aldolase. Eur J Biochem. 1970 Aug;15(2):360–366. doi: 10.1111/j.1432-1033.1970.tb01016.x. [DOI] [PubMed] [Google Scholar]
  7. Arnold H., Pette D. Binding of glycolytic enzymes to structure proteins of the muscle. Eur J Biochem. 1968 Nov;6(2):163–171. doi: 10.1111/j.1432-1033.1968.tb00434.x. [DOI] [PubMed] [Google Scholar]
  8. Brooks S. P., Storey K. B. Where is the glycolytic complex? A critical evaluation of present data from muscle tissue. FEBS Lett. 1991 Jan 28;278(2):135–138. doi: 10.1016/0014-5793(91)80101-8. [DOI] [PubMed] [Google Scholar]
  9. Clarke F. M., Masters C. J. On the association of glycolytic enzymes with structural proteins of skeletal muscle. Biochim Biophys Acta. 1975 Jan 13;381(1):37–46. doi: 10.1016/0304-4165(75)90187-7. [DOI] [PubMed] [Google Scholar]
  10. Clarke F. M., Masters C. J. On the distribution of aldolase isoenzymes in subcellular fractions from rat brain. Arch Biochem Biophys. 1973 Jun;156(2):673–683. doi: 10.1016/0003-9861(73)90320-2. [DOI] [PubMed] [Google Scholar]
  11. Clarke F. M., Masters C. J. On the reversible and selective adsorption of aldolase isoenzymes in rat brain. Arch Biochem Biophys. 1972 Nov;153(1):258–265. doi: 10.1016/0003-9861(72)90444-4. [DOI] [PubMed] [Google Scholar]
  12. Clegg J. S. Properties and metabolism of the aqueous cytoplasm and its boundaries. Am J Physiol. 1984 Feb;246(2 Pt 2):R133–R151. doi: 10.1152/ajpregu.1984.246.2.R133. [DOI] [PubMed] [Google Scholar]
  13. Clifton P. M., Chang L., Mackinnon A. M. Development of an automated Lowry protein assay for the Cobas-Bio centrifugal analyzer. Anal Biochem. 1988 Jul;172(1):165–168. doi: 10.1016/0003-2697(88)90426-5. [DOI] [PubMed] [Google Scholar]
  14. Davies D. R., Detheux M., Van Schaftingen E. Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes. Eur J Biochem. 1990 Sep 11;192(2):283–289. doi: 10.1111/j.1432-1033.1990.tb19225.x. [DOI] [PubMed] [Google Scholar]
  15. Eriksson A. M., Zetterqvist M. A., Lundgren B., Andersson K., Beije B., DePierre J. W. Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin-permeabilization of hepatocytes isolated from control and clofibrate-treated mice. Eur J Biochem. 1991 Jun 1;198(2):471–476. doi: 10.1111/j.1432-1033.1991.tb16037.x. [DOI] [PubMed] [Google Scholar]
  16. Foemmel R. S., Gray R. H., Bernstein I. A. Intracellular localization of fructose 1,6-bisphosphate aldolase. J Biol Chem. 1975 Mar 10;250(5):1892–1897. [PubMed] [Google Scholar]
  17. Hers H. G., Hue L. Gluconeogenesis and related aspects of glycolysis. Annu Rev Biochem. 1983;52:617–653. doi: 10.1146/annurev.bi.52.070183.003153. [DOI] [PubMed] [Google Scholar]
  18. Jenkins J. D., Madden D. P., Steck T. L. Association of phosphofructokinase and aldolase with the membrane of the intact erythrocyte. J Biol Chem. 1984 Aug 10;259(15):9374–9378. [PubMed] [Google Scholar]
  19. Keleti T., Ovádi J., Batke J. Kinetic and physico-chemical analysis of enzyme complexes and their possible role in the control of metabolism. Prog Biophys Mol Biol. 1989;53(2):105–152. doi: 10.1016/0079-6107(89)90016-3. [DOI] [PubMed] [Google Scholar]
  20. Kellermayer M., Ludany A., Jobst K., Szucs G., Trombitas K., Hazlewood C. F. Cocompartmentation of proteins and K+ within the living cell. Proc Natl Acad Sci U S A. 1986 Feb;83(4):1011–1015. doi: 10.1073/pnas.83.4.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Knull H. R., Walsh J. L. Association of glycolytic enzymes with the cytoskeleton. Curr Top Cell Regul. 1992;33:15–30. doi: 10.1016/b978-0-12-152833-1.50007-1. [DOI] [PubMed] [Google Scholar]
  22. Lebherz H. G., Rutter W. J. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry. 1969 Jan;8(1):109–121. doi: 10.1021/bi00829a016. [DOI] [PubMed] [Google Scholar]
  23. Neuzil J., Danielson H., Welch G. R., Ovádi J. Cooperative effect of fructose bisphosphate and glyceraldehyde-3-phosphate dehydrogenase on aldolase action. Biochim Biophys Acta. 1990 Mar 1;1037(3):307–312. doi: 10.1016/0167-4838(90)90030-j. [DOI] [PubMed] [Google Scholar]
  24. Ottaway J. H., Mowbray J. The role of compartmentation in the control of glycolysis. Curr Top Cell Regul. 1977;12:107–208. doi: 10.1016/b978-0-12-152812-6.50010-x. [DOI] [PubMed] [Google Scholar]
  25. Pagliaro L., Taylor D. L. 2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells. J Cell Biol. 1992 Aug;118(4):859–863. doi: 10.1083/jcb.118.4.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pagliaro L., Taylor D. L. Aldolase exists in both the fluid and solid phases of cytoplasm. J Cell Biol. 1988 Sep;107(3):981–991. doi: 10.1083/jcb.107.3.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. ROODYN D. B. The binding of aldolase to isolated nuclei. Biochim Biophys Acta. 1957 Jul;25(1):129–131. doi: 10.1016/0006-3002(57)90427-4. [DOI] [PubMed] [Google Scholar]
  28. Schliwa M., van Blerkom J., Porter K. R. Stabilization and the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4329–4333. doi: 10.1073/pnas.78.7.4329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seeholzer S. H. Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1237–1241. doi: 10.1073/pnas.90.4.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Srere P. A. Complexes of sequential metabolic enzymes. Annu Rev Biochem. 1987;56:89–124. doi: 10.1146/annurev.bi.56.070187.000513. [DOI] [PubMed] [Google Scholar]
  31. Stanton R. C., Seifter J. L., Boxer D. C., Zimmerman E., Cantley L. C. Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity. J Biol Chem. 1991 Jul 5;266(19):12442–12448. [PubMed] [Google Scholar]
  32. Tian W. N., Pignatare J. N., Stanton R. C. Signal transduction proteins that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized cells. J Biol Chem. 1994 May 20;269(20):14798–14805. [PubMed] [Google Scholar]
  33. Uyeda K. Interactions of glycolytic enzymes with cellular membranes. Curr Top Cell Regul. 1992;33:31–46. doi: 10.1016/b978-0-12-152833-1.50008-3. [DOI] [PubMed] [Google Scholar]
  34. Vértessy B. G., Orosz F., Ovádi J. Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates. Biochim Biophys Acta. 1991 Jun 24;1078(2):236–242. doi: 10.1016/0167-4838(91)90564-g. [DOI] [PubMed] [Google Scholar]
  35. Walsh J. L., Keith T. J., Knull H. R. Glycolytic enzyme interactions with tubulin and microtubules. Biochim Biophys Acta. 1989 Nov 9;999(1):64–70. doi: 10.1016/0167-4838(89)90031-9. [DOI] [PubMed] [Google Scholar]
  36. Walsh J. L., Knull H. R. Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol). Biochim Biophys Acta. 1988 Jan 4;952(1):83–91. doi: 10.1016/0167-4838(88)90104-5. [DOI] [PubMed] [Google Scholar]
  37. Weiss T. L., Zieske J. D., Bernstein I. A. Reversible microsomal binding of hepatic aldolase. Biochim Biophys Acta. 1981 Oct 13;661(2):221–229. doi: 10.1016/0005-2744(81)90007-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES