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. 1995 Oct;96(4):1927–1935. doi: 10.1172/JCI118238

Influence of glutathione S-transferase B (ligandin) on the intermembrane transfer of bilirubin. Implications for the intracellular transport of nonsubstrate ligands in hepatocytes.

S D Zucker 1, W Goessling 1, B J Ransil 1, J L Gollan 1
PMCID: PMC185829  PMID: 7560084

Abstract

To examine the hypothesis that glutathione S-transferases (GST) play an important role in the hepatocellular transport of hydrophobic organic anions, the kinetics of the spontaneous transfer of unconjugated bilirubin between membrane vesicles and rat liver glutathione S-transferase B (ligandin) was studied, using stopped-flow fluorometry. Bilirubin transfer from glutathione S-transferase B to phosphatidylcholine vesicles was best described by a single exponential function, with a rate constant of 8.0 +/- 0.7 s-1 (+/- SD) at 25 degrees C. The variations in transfer rate with respect to acceptor phospholipid concentration provide strong evidence for aqueous diffusion of free bilirubin. This finding was verified using rhodamine-labeled microsomal membranes as acceptors. Bilirubin transfer from phospholipid vesicles to GST also exhibited diffusional kinetics. Thermodynamic parameters for bilirubin dissociation from GST were similar to those for human serum albumin. The rate of bilirubin transfer from rat liver basolateral plasma membranes to acceptor vesicles in the presence of glutathione S-transferase B declined asymptotically with increasing GST concentration. These data suggest that glutathione S-transferase B does not function as an intracellular bilirubin transporter, although expression of this protein may serve to regulate the delivery of bilirubin, and other nonsubstrate ligands, to sites of metabolism within the cell.

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Selected References

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  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. Barenholz Y., Gibbes D., Litman B. J., Goll J., Thompson T. E., Carlson R. D. A simple method for the preparation of homogeneous phospholipid vesicles. Biochemistry. 1977 Jun 14;16(12):2806–2810. doi: 10.1021/bi00631a035. [DOI] [PubMed] [Google Scholar]
  3. Bennett C. F., Yeoman L. C. Microinjected glutathione S-transferase Yb subunits translocate to the cell nucleus. Biochem J. 1987 Oct 1;247(1):109–112. doi: 10.1042/bj2470109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhargava M. M., Ohmi N., Listowsky I., Arias I. M. Subunit composition, organic anion binding, catalytic and immunological properties of ligandin from rat testis. J Biol Chem. 1980 Jan 25;255(2):724–727. [PubMed] [Google Scholar]
  5. Boyer T. D., Olsen E. Role of glutathione S-transferases in heme transport. Biochem Pharmacol. 1991 Jun 21;42(1):188–190. doi: 10.1016/0006-2952(91)90699-6. [DOI] [PubMed] [Google Scholar]
  6. Boyer T. D. The glutathione S-transferases: an update. Hepatology. 1989 Mar;9(3):486–496. doi: 10.1002/hep.1840090324. [DOI] [PubMed] [Google Scholar]
  7. Boyer T. D., Vessey D. A., Holcomb C., Saley N. Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferases. Biochem J. 1984 Jan 1;217(1):179–185. doi: 10.1042/bj2170179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boyer T. D., Zakim D., Vessey D. A. Do the soluble glutathione S-transferases have direct access to membrane-bound substrates? Biochem Pharmacol. 1983 Jan 1;32(1):29–35. doi: 10.1016/0006-2952(83)90647-0. [DOI] [PubMed] [Google Scholar]
  9. Caccuri A. M., Aceto A., Piemonte F., Di Ilio C., Rosato N., Federici G. Interaction of hemin with placental glutathione transferase. Eur J Biochem. 1990 May 20;189(3):493–497. doi: 10.1111/j.1432-1033.1990.tb15514.x. [DOI] [PubMed] [Google Scholar]
  10. Carmagnol F., Sinet P. M., Rapin J., Jerome H. Glutathione-S-transferase of human red blood cells; assay, values in normal subjects and in two pathological circumstances: hyperbilirubinemia and impaired renal function. Clin Chim Acta. 1981 Dec 9;117(2):209–217. doi: 10.1016/0009-8981(81)90040-1. [DOI] [PubMed] [Google Scholar]
  11. Cu A., Bellah G. G., Lightner D. A. Letter: On the fluorescence of bilirubin. J Am Chem Soc. 1975 Apr 30;97(9):2579–2580. doi: 10.1021/ja00842a066. [DOI] [PubMed] [Google Scholar]
  12. Fleischner G., Robbins J., Arias I. M. Immunological studies of Y protein. A major cytoplasmic organic anion-binding protein in rat liver. J Clin Invest. 1972 Mar;51(3):677–684. doi: 10.1172/JCI106856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gadella T. W., Jr, Wirtz K. W. Phospholipid binding and transfer by the nonspecific lipid-transfer protein (sterol carrier protein 2). A kinetic model. Eur J Biochem. 1994 Mar 15;220(3):1019–1028. doi: 10.1111/j.1432-1033.1994.tb18707.x. [DOI] [PubMed] [Google Scholar]
  14. Habig W. H., Jakoby W. B. Assays for differentiation of glutathione S-transferases. Methods Enzymol. 1981;77:398–405. doi: 10.1016/s0076-6879(81)77053-8. [DOI] [PubMed] [Google Scholar]
  15. Hahm J. S., Ostrow J. D., Mukerjee P., Celic L. Ionization and self-association of unconjugated bilirubin, determined by rapid solvent partition from chloroform, with further studies of bilirubin solubility. J Lipid Res. 1992 Aug;33(8):1123–1137. [PubMed] [Google Scholar]
  16. Husby P., Srai K. S., Ketterer B., Romslo I. Effect of ligandin on the efflux of Co-deuteroporphyrin from isolated rat liver mitochondria. Biochem Biophys Res Commun. 1981 May 29;100(2):651–659. doi: 10.1016/s0006-291x(81)80225-2. [DOI] [PubMed] [Google Scholar]
  17. Jagt D. L., Wilson S. P., Dean V. L., Simons P. C. Bilirubin binding to rat liver ligandins (glutathione S-transferases A and B). Relationship between bilirubin binding and transferase activity. J Biol Chem. 1982 Feb 25;257(4):1997–2001. [PubMed] [Google Scholar]
  18. Kamisaka K., Habig W. H., Ketley J. N., Arias M., Jakoby W. B. Multiple forms of human glutathione S-transferase and their affinity for bilirubin. Eur J Biochem. 1975 Dec 1;60(1):153–161. doi: 10.1111/j.1432-1033.1975.tb20987.x. [DOI] [PubMed] [Google Scholar]
  19. Kamisaka K., Listowsky I., Gatmaitan Z., Arias I. M. Interactions of bilirubin and other ligands with ligandin. Biochemistry. 1975 May 20;14(10):2175–2180. doi: 10.1021/bi00681a021. [DOI] [PubMed] [Google Scholar]
  20. Kaplowitz N. Physiological significance of glutathione S-transferases. Am J Physiol. 1980 Dec;239(6):G439–G444. doi: 10.1152/ajpgi.1980.239.6.G439. [DOI] [PubMed] [Google Scholar]
  21. Ketley J. N., Habig W. H., Jakoby W. B. Binding of nonsubstrate ligands to the glutathione S-transferases. J Biol Chem. 1975 Nov 25;250(22):8670–8673. [PubMed] [Google Scholar]
  22. Kim H. K., Storch J. Free fatty acid transfer from rat liver fatty acid-binding protein to phospholipid vesicles. Effect of ligand and solution properties. J Biol Chem. 1992 Jan 5;267(1):77–82. [PubMed] [Google Scholar]
  23. Kim H. K., Storch J. Mechanism of free fatty acid transfer from rat heart fatty acid-binding protein to phospholipid membranes. Evidence for a collisional process. J Biol Chem. 1992 Oct 5;267(28):20051–20056. [PubMed] [Google Scholar]
  24. Kirsch R., Fleischner G., Kamisaka K., Arias I. M. Structural and functional studies of ligandin, a major renal organic anion-binding protein. J Clin Invest. 1975 May;55(5):1009–1019. doi: 10.1172/JCI108001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LeBlanc G. A. Hepatic vectorial transport of xenobiotics. Chem Biol Interact. 1994 Feb;90(2):101–120. doi: 10.1016/0009-2797(94)90097-3. [DOI] [PubMed] [Google Scholar]
  26. Levi A. J., Gatmaitan Z., Arias I. M. Deficiency of hepatic organic anion-binding protein, impaired organic amnion uptake by liver and "physiologic" jaundice in newborn monkeys. N Engl J Med. 1970 Nov 19;283(21):1136–1139. doi: 10.1056/NEJM197011192832104. [DOI] [PubMed] [Google Scholar]
  27. Levi A. J., Gatmaitan Z., Arias I. M. Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions. J Clin Invest. 1969 Nov;48(11):2156–2167. doi: 10.1172/JCI106182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Leys E. J., Crouse G. F., Kellems R. E. Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus. J Cell Biol. 1984 Jul;99(1 Pt 1):180–187. doi: 10.1083/jcb.99.1.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liem H. H., Grasso J. A., Vincent S. H., Muller-Eberhard U. Protein-mediated efflux of heme from isolated rat liver mitochondria. Biochem Biophys Res Commun. 1990 Mar 16;167(2):528–534. doi: 10.1016/0006-291x(90)92056-6. [DOI] [PubMed] [Google Scholar]
  30. Listowsky I., Abramovitz M., Homma H., Niitsu Y. Intracellular binding and transport of hormones and xenobiotics by glutathione-S-transferases. Drug Metab Rev. 1988;19(3-4):305–318. doi: 10.3109/03602538808994138. [DOI] [PubMed] [Google Scholar]
  31. Luby-Phelps K., Castle P. E., Taylor D. L., Lanni F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4910–4913. doi: 10.1073/pnas.84.14.4910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McCormack M., Brecher P. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes. Biochem J. 1987 Jun 15;244(3):717–723. doi: 10.1042/bj2440717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meier P. J., Boyer J. L. Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes. Methods Enzymol. 1990;192:534–545. doi: 10.1016/0076-6879(90)92092-r. [DOI] [PubMed] [Google Scholar]
  34. Nichols J. W. Kinetics of fluorescent-labeled phosphatidylcholine transfer between nonspecific lipid transfer protein and phospholipid vesicles. Biochemistry. 1988 Mar 22;27(6):1889–1896. doi: 10.1021/bi00406a014. [DOI] [PubMed] [Google Scholar]
  35. Nichols J. W., Pagano R. E. Resonance energy transfer assay of protein-mediated lipid transfer between vesicles. J Biol Chem. 1983 May 10;258(9):5368–5371. [PubMed] [Google Scholar]
  36. Nichols J. W., Pagano R. E. Use of resonance energy transfer to study the kinetics of amphiphile transfer between vesicles. Biochemistry. 1982 Apr 13;21(8):1720–1726. doi: 10.1021/bi00537a003. [DOI] [PubMed] [Google Scholar]
  37. Nishihira J., Ishibashi T., Sakai M., Tsuda S., Hikichi K. Identification of the hydrophobic ligand-binding region in recombinant glutathione S-transferase P and its binding effect on the conformational state of the enzyme. Arch Biochem Biophys. 1993 Apr;302(1):128–133. doi: 10.1006/abbi.1993.1190. [DOI] [PubMed] [Google Scholar]
  38. Parola M., Biocca M. E., Leonarduzzi G., Albano E., Dianzani M. U., Gilmore K. S., Meyer D. J., Ketterer B., Slater T. F., Cheeseman K. H. Constitutive and inducible profile of glutathione S-transferase subunits in biliary epithelial cells and hepatocytes isolated from rat liver. Biochem J. 1993 Apr 15;291(Pt 2):641–647. doi: 10.1042/bj2910641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pickett C. B., Lu A. Y. Glutathione S-transferases: gene structure, regulation, and biological function. Annu Rev Biochem. 1989;58:743–764. doi: 10.1146/annurev.bi.58.070189.003523. [DOI] [PubMed] [Google Scholar]
  40. Runquist E. A., Helmkamp G. M., Jr Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein. Biochim Biophys Acta. 1988 May 9;940(1):21–32. doi: 10.1016/0005-2736(88)90004-1. [DOI] [PubMed] [Google Scholar]
  41. Rushmore T. H., Pickett C. B. Glutathione S-transferases, structure, regulation, and therapeutic implications. J Biol Chem. 1993 Jun 5;268(16):11475–11478. [PubMed] [Google Scholar]
  42. Scharschmidt B. F., Keeffe E. B., Blankenship N. M., Ockner R. K. Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity. J Lab Clin Med. 1979 May;93(5):790–799. [PubMed] [Google Scholar]
  43. Senjo M., Ishibashi T., Imai Y. Purification and characterization of cytosolic liver protein facilitating heme transport into apocytochrome b5 from mitochondria. Evidence for identifying the heme transfer protein as belonging to a group of glutathione S-transferases. J Biol Chem. 1985 Aug 5;260(16):9191–9196. [PubMed] [Google Scholar]
  44. Simons P. C., Jagt D. L. Bilirubin binding to human liver ligandin (glutathione S-transferase). J Biol Chem. 1980 May 25;255(10):4740–4744. [PubMed] [Google Scholar]
  45. Spivak W., Yuey W. Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease. Biochem J. 1986 Feb 15;234(1):101–109. doi: 10.1042/bj2340101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Storch J., Bass N. M. Transfer of fluorescent fatty acids from liver and heart fatty acid-binding proteins to model membranes. J Biol Chem. 1990 May 15;265(14):7827–7831. [PubMed] [Google Scholar]
  47. Stubbs C. D., Kinosita K., Jr, Munkonge F., Quinn P. J., Ikegami A. The dynamics of lipid motion in sarcoplasmic reticulum membranes determined by steady-state and time-resolved fluorescence measurements on 1,6-diphenyl-1,3,5-hexatriene and related molecules. Biochim Biophys Acta. 1984 Sep 5;775(3):374–380. doi: 10.1016/0005-2736(84)90193-7. [DOI] [PubMed] [Google Scholar]
  48. Sugiyama Y., Sugimoto M., Stolz A., Kaplowitz N. Comparison of the binding affinities of five forms of rat glutathione S-transferases for bilirubin, sulfobromophthalein and hematin. Biochem Pharmacol. 1984 Nov 1;33(21):3511–3513. doi: 10.1016/0006-2952(84)90128-x. [DOI] [PubMed] [Google Scholar]
  49. Theilmann L., Stollman Y. R., Arias I. M., Wolkoff A. W. Does Z-protein have a role in transport of bilirubin and bromosulfophthalein by isolated perfused rat liver? Hepatology. 1984 Sep-Oct;4(5):923–926. doi: 10.1002/hep.1840040523. [DOI] [PubMed] [Google Scholar]
  50. Tipping E., Ketterer B., Christodoulides L., Enderby G. The non-convalent binding of small molecules by ligandin. Interactions with steroids and their conjugates, fatty acids, bromosulphophthalein carcinogens, glutathione and realted compounds. Eur J Biochem. 1976 Aug 16;67(2):583–590. doi: 10.1111/j.1432-1033.1976.tb10724.x. [DOI] [PubMed] [Google Scholar]
  51. Tipping E., Ketterer B., Christodoulides L. Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some organic anions (bromosulphophthalein, oestrone sulphate, haem and bilirubin) that bind to ligandin and aminoazo-dye-binding protein A. Biochem J. 1979 May 15;180(2):327–337. doi: 10.1042/bj1800327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tipping E., Ketterer B. The influence of soluble binding proteins on lipophile transport and metabolism in hepatocytes. Biochem J. 1981 May 1;195(2):441–452. doi: 10.1042/bj1950441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Trotter P. J., Voelker D. R. Lipid transport processes in eukaryotic cells. Biochim Biophys Acta. 1994 Aug 4;1213(3):241–262. doi: 10.1016/0005-2760(94)00073-5. [DOI] [PubMed] [Google Scholar]
  54. Vessey D. A., Boyer T. D. Characterization of the activation of rat liver glutathione S-transferases by nonsubstrate ligands. Toxicol Appl Pharmacol. 1988 Apr;93(2):275–280. doi: 10.1016/0041-008x(88)90127-5. [DOI] [PubMed] [Google Scholar]
  55. Vos R. M., Van Bladeren P. J. Glutathione S-transferases in relation to their role in the biotransformation of xenobiotics. Chem Biol Interact. 1990;75(3):241–265. doi: 10.1016/0009-2797(90)90069-y. [DOI] [PubMed] [Google Scholar]
  56. Whitmer D. I., Ziurys J. C., Gollan J. L. Hepatic microsomal glucuronidation of bilirubin in unilamellar liposomal membranes. Implications for intracellular transport of lipophilic substrates. J Biol Chem. 1984 Oct 10;259(19):11969–11975. [PubMed] [Google Scholar]
  57. Wolkoff A. W., Goresky C. A., Sellin J., Gatmaitan Z., Arias I. M. Role of ligandin in transfer of bilirubin from plasma into liver. Am J Physiol. 1979 Jun;236(6):E638–E648. doi: 10.1152/ajpendo.1979.236.6.E638. [DOI] [PubMed] [Google Scholar]
  58. Wootan M. G., Bernlohr D. A., Storch J. Mechanism of fluorescent fatty acid transfer from adipocyte fatty acid binding protein to membranes. Biochemistry. 1993 Aug 24;32(33):8622–8627. doi: 10.1021/bi00084a033. [DOI] [PubMed] [Google Scholar]
  59. Zucker S. D., Goessling W., Gollan J. L. Kinetics of bilirubin transfer between serum albumin and membrane vesicles. Insight into the mechanism of organic anion delivery to the hepatocyte plasma membrane. J Biol Chem. 1995 Jan 20;270(3):1074–1081. doi: 10.1074/jbc.270.3.1074. [DOI] [PubMed] [Google Scholar]
  60. Zucker S. D., Goessling W., Zeidel M. L., Gollan J. L. Membrane lipid composition and vesicle size modulate bilirubin intermembrane transfer. Evidence for membrane-directed trafficking of bilirubin in the hepatocyte. J Biol Chem. 1994 Jul 29;269(30):19262–19270. [PubMed] [Google Scholar]
  61. Zucker S. D., Storch J., Zeidel M. L., Gollan J. L. Mechanism of the spontaneous transfer of unconjugated bilirubin between small unilamellar phosphatidylcholine vesicles. Biochemistry. 1992 Mar 31;31(12):3184–3192. doi: 10.1021/bi00127a020. [DOI] [PubMed] [Google Scholar]
  62. van Ommen B., Bogaards J. J., Peters W. H., Blaauboer B., van Bladeren P. J. Quantification of human hepatic glutathione S-transferases. Biochem J. 1990 Aug 1;269(3):609–613. doi: 10.1042/bj2690609. [DOI] [PMC free article] [PubMed] [Google Scholar]

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