Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 Dec 1;240(2):549–557. doi: 10.1042/bj2400549

Characterization of the binding of human low-density lipoprotein to primary monolayer cultures of rat hepatocytes.

A M Salter, J Saxton, D N Brindley
PMCID: PMC1147449  PMID: 2434075

Abstract

The binding of human low-density lipoprotein labelled with 125I to rat hepatocytes in monolayer culture was measured at 4 degrees C. Evidence for two different specific binding sites was obtained. Binding to Site 1 was characterized by: being displaced by dextran sulphate or heparin; being dependent on Ca2+; having a Kd value of about 15 micrograms of protein/ml; not being significantly displaced by a 20-fold excess unlabelled low-density lipoprotein that had been reductively methylated; being displaced by approx. 40% by a 20-fold protein excess of unlabelled human high-density lipoprotein, HDL3, and increasing with time in culture when newborn-calf serum was present in the medium. The binding to Site 2 had the following properties: it was not displaced by sulphated polysaccharides; it was only partially Ca2+-dependent, and the presence of EDTA increased the Kd value; the apparent Kd value in the presence of Ca2+ was approx. 30 micrograms of protein/ml, which was significantly higher than for Site 1; it was displaced by approx. 30% with a 20-fold excess of low-density lipoprotein that had been methylated; it was displaced by unlabelled HDL3 to a similar extent to Site 1; it did not increase significantly with time in culture. The characteristics of binding to Sites 1 and 2 are discussed in relation to the receptors for low-density lipoproteins that have previously been described in various cell types. It is proposed that the experimental system described in this paper is suitable for studying the regulation of the binding of low-density lipoproteins to hepatocytes.

Full text

PDF
549

Selected References

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

  1. Bachorik P. S., Franklin F. A., Virgil D. G., Kwiterovich P. O., Jr High-affinity uptake and degradation of apolipoprotein E free high-density lipoprotein and low-density lipoprotein in cultured porcine hepatocytes. Biochemistry. 1982 Oct 26;21(22):5675–5684. doi: 10.1021/bi00265a044. [DOI] [PubMed] [Google Scholar]
  2. Bachorik P. S., Kwiterovich P. O., Cooke J. C. Isolation of a porcine liver plasma membrane fraction that binds low density lipoproteins. Biochemistry. 1978 Nov 28;17(24):5287–5299. doi: 10.1021/bi00617a032. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cascales C., Mangiapane E. H., Brindley D. N. Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes. Biochem J. 1984 May 1;219(3):911–916. doi: 10.1042/bj2190911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chao Y. S., Windler E. E., Chen G. C., Havel R. J. Hepatic catabolism of rat and human lipoproteins in rats treated with 17 alpha-ethinyl estradiol. J Biol Chem. 1979 Nov 25;254(22):11360–11366. [PubMed] [Google Scholar]
  6. Chao Y., Yamin T. T., Alberts A. W. Effects of cholestyramine on low density lipoprotein binding sites on liver membranes from rabbits with endogenous hypercholesterolemia induced by a wheat starch-casein diet. J Biol Chem. 1982 Apr 10;257(7):3623–3627. [PubMed] [Google Scholar]
  7. Daniel T. O., Schneider W. J., Goldstein J. L., Brown M. S. Visualization of lipoprotein receptors by ligand blotting. J Biol Chem. 1983 Apr 10;258(7):4606–4611. [PubMed] [Google Scholar]
  8. Drevon C. A., Attie A. D., Pangburn S. H., Steinberg D. Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts. J Lipid Res. 1981 Jan;22(1):37–46. [PubMed] [Google Scholar]
  9. Edge S. B., Hoeg J. M., Triche T., Schneider P. D., Brewer H. B., Jr Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor. J Biol Chem. 1986 Mar 15;261(8):3800–3806. [PubMed] [Google Scholar]
  10. Goldstein J. L., Basu S. K., Brunschede G. Y., Brown M. S. Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans. Cell. 1976 Jan;7(1):85–95. doi: 10.1016/0092-8674(76)90258-0. [DOI] [PubMed] [Google Scholar]
  11. Goldstein J. L., Brown M. S. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. doi: 10.1146/annurev.bi.46.070177.004341. [DOI] [PubMed] [Google Scholar]
  12. HAVEL R. J., EDER H. A., BRAGDON J. H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955 Sep;34(9):1345–1353. doi: 10.1172/JCI103182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Habeeb A. F. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem. 1966 Mar;14(3):328–336. doi: 10.1016/0003-2697(66)90275-2. [DOI] [PubMed] [Google Scholar]
  14. Harkes L., van Berkel T. J. A saturable, high-affinity binding site for human low density lipoprotein on freshly isolated rat hepatocytes. Biochim Biophys Acta. 1982 Sep 14;712(3):677–683. doi: 10.1016/0005-2760(82)90297-1. [DOI] [PubMed] [Google Scholar]
  15. Havekes L. M., Schouten D., de Wit E. C., Cohen L. H., Griffioen M., van Hinsbergh V. W., Princen H. M. Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions. Biochim Biophys Acta. 1986 Feb 12;875(2):236–246. doi: 10.1016/0005-2760(86)90173-6. [DOI] [PubMed] [Google Scholar]
  16. Havel R. J. George Lyman Duff memorial lecture. Role of the liver in atherosclerosis. Arteriosclerosis. 1985 Nov-Dec;5(6):569–580. doi: 10.1161/01.atv.5.6.569. [DOI] [PubMed] [Google Scholar]
  17. Hawrot E., Patterson P. H. Long-term culture of dissociated sympathetic neurons. Methods Enzymol. 1979;58:574–584. doi: 10.1016/s0076-6879(79)58174-9. [DOI] [PubMed] [Google Scholar]
  18. Hoeg J. M., Demosky S. J., Jr, Schaefer E. J., Starzl T. E., Brewer H. B., Jr Characterization of hepatic low density lipoprotein binding and cholesterol metabolism in normal and homozygous familial hypercholesterolemic subjects. J Clin Invest. 1984 Feb;73(2):429–436. doi: 10.1172/JCI111229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hui D. Y., Innerarity T. L., Mahley R. W. Lipoprotein binding to canine hepatic membranes. Metabolically distinct apo-E and apo-B,E receptors. J Biol Chem. 1981 Jun 10;256(11):5646–5655. [PubMed] [Google Scholar]
  20. Innerarity T. L., Pitas R. E., Mahley R. W. Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity. J Biol Chem. 1980 Dec 10;255(23):11163–11172. [PubMed] [Google Scholar]
  21. Jones A. L., Hradek G. T., Hornick C., Renaud G., Windler E. E., Havel R. J. Uptake and processing of remnants of chylomicrons and very low density lipoproteins by rat liver. J Lipid Res. 1984 Nov;25(11):1151–1158. [PubMed] [Google Scholar]
  22. Kovanen P. T., Brown M. S., Goldstein J. L. Increased binding of low density lipoprotein to liver membranes from rats treated with 17 alpha-ethinyl estradiol. J Biol Chem. 1979 Nov 25;254(22):11367–11373. [PubMed] [Google Scholar]
  23. Lopes-Virella M. F., Stone P., Ellis S., Colwell J. A. Cholesterol determination in high-density lipoproteins separated by three different methods. Clin Chem. 1977 May;23(5):882–884. [PubMed] [Google Scholar]
  24. Mahley R. W., Hui D. Y., Innerarity T. L., Weisgraber K. H. Two independent lipoprotein receptors on hepatic membranes of dog, swine, and man. Apo-B,E and apo-E receptors. J Clin Invest. 1981 Nov;68(5):1197–1206. doi: 10.1172/JCI110365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mahley R. W., Weisgraber K. H., Innerarity T. L. Interaction of plasma lipoproteins containing apolipoproteins B and E with heparin and cell surface receptors. Biochim Biophys Acta. 1979 Oct 26;575(1):81–91. doi: 10.1016/0005-2760(79)90133-4. [DOI] [PubMed] [Google Scholar]
  26. Mahley R. W., Weisgraber K. H., Melchior G. W., Innerarity T. L., Holcombe K. S. Inhibition of receptor-mediated clearance of lysine and arginine-modified lipoproteins from the plasma of rats and monkeys. Proc Natl Acad Sci U S A. 1980 Jan;77(1):225–229. doi: 10.1073/pnas.77.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martin-Sanz P., Hopewell R., Brindley D. N. Spermine promotes the translocation of phosphatidate phosphohydrolase from the cytosol to the microsomal fraction of rat liver and it enhances the effects of oleate in this respect. FEBS Lett. 1985 Jan 7;179(2):262–266. doi: 10.1016/0014-5793(85)80531-7. [DOI] [PubMed] [Google Scholar]
  28. McFARLANE A. S. Efficient trace-labelling of proteins with iodine. Nature. 1958 Jul 5;182(4627):53–53. doi: 10.1038/182053a0. [DOI] [PubMed] [Google Scholar]
  29. Oram J. F., Albers J. J., Cheung M. C., Bierman E. L. The effects of subfractions of high density lipoprotein on cholesterol efflux from cultured fibroblasts. Regulation of low density lipoprotein receptor activity. J Biol Chem. 1981 Aug 25;256(16):8348–8356. [PubMed] [Google Scholar]
  30. Oram J. F. Effects of high density lipoprotein subfractions on cholesterol homeostasis in human fibroblasts and arterial smooth muscle cells. Arteriosclerosis. 1983 Sep-Oct;3(5):420–432. doi: 10.1161/01.atv.3.5.420. [DOI] [PubMed] [Google Scholar]
  31. Ose L., Ose T., Norum K. R., Berg T. Uptake and degradation of 125I-labelled high density lipoproteins in rat liver cells in vivo and in vitro. Biochim Biophys Acta. 1979 Sep 28;574(3):521–536. doi: 10.1016/0005-2760(79)90248-0. [DOI] [PubMed] [Google Scholar]
  32. Ose T., Berg T., Norum K. R., Ose L. Catabolism of (125-I)low density lipoproteins in isolated rat liver cells. Biochem Biophys Res Commun. 1980 Nov 17;97(1):192–199. doi: 10.1016/s0006-291x(80)80153-7. [DOI] [PubMed] [Google Scholar]
  33. Pittman R. C., Attie A. D., Carew T. E., Steinberg D. Tissue sites of degradation of low density lipoprotein: application of a method for determining the fate of plasma proteins. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5345–5349. doi: 10.1073/pnas.76.10.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pittner R. A., Fears R., Brindley D. N. Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis. Biochem J. 1985 Jan 15;225(2):455–462. doi: 10.1042/bj2250455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roach P. D., Noël S. P. Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver. J Lipid Res. 1985 Jun;26(6):713–720. [PubMed] [Google Scholar]
  36. Schneider W. J., Beisiegel U., Goldstein J. L., Brown M. S. Purification of the low density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J Biol Chem. 1982 Mar 10;257(5):2664–2673. [PubMed] [Google Scholar]
  37. Shepherd J., Bedford D. K., Morgan H. G. Radioiodination of human low density lipoprotein: a comparison of four methods. Clin Chim Acta. 1976 Jan 2;66(1):97–109. doi: 10.1016/0009-8981(76)90376-4. [DOI] [PubMed] [Google Scholar]
  38. Soltys P. A., Portman O. W. Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes. Biochim Biophys Acta. 1979 Sep 28;574(3):505–520. doi: 10.1016/0005-2760(79)90247-9. [DOI] [PubMed] [Google Scholar]
  39. Tamai T., Patsch W., Lock D., Schonfeld G. Receptors for homologous plasma lipoproteins on a rat hepatoma cell line. J Lipid Res. 1983 Dec;24(12):1568–1577. [PubMed] [Google Scholar]
  40. Van Berkel T. J., Kruijt J. K., Van Gent T., Van Tol A. Saturable high affinity binding of low density and high density lipoprotein by parenchymal and non-parenchymal cells from rat liver. Biochem Biophys Res Commun. 1980 Feb 12;92(3):1002–1008. doi: 10.1016/0006-291x(80)90801-3. [DOI] [PubMed] [Google Scholar]
  41. Van Tol A., Van 't Hooft F. M., Van Gent T. Discrepancies in the catabolic pathways of rat and human low density lipoproteins as revealed by partial hepatectomy in the rat. Atherosclerosis. 1978 Apr;29(4):449–457. doi: 10.1016/0021-9150(78)90173-9. [DOI] [PubMed] [Google Scholar]
  42. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  43. Weisgraber K. H., Innerarity T. L., Mahley R. W. Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts. J Biol Chem. 1978 Dec 25;253(24):9053–9062. [PubMed] [Google Scholar]
  44. Weisgraber K. H., Mahley R. W. Subfractionation of human high density lipoproteins by heparin-Sepharose affinity chromatography. J Lipid Res. 1980 Mar;21(3):316–325. [PubMed] [Google Scholar]
  45. Windler E. E., Kovanen P. T., Chao Y. S., Brown M. S., Havel R. J., Goldstein J. L. The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E. J Biol Chem. 1980 Nov 10;255(21):10464–10471. [PubMed] [Google Scholar]

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

RESOURCES