Logo of biochemjBJ Latest papers and much more!
Biochem J. 1997 May 1; 323(Pt 3): 649–659.
PMCID: PMC1218367

Kinetics of low-density lipoprotein receptor activity in Hep-G2 cells: derivation and validation of a Briggs-Haldane-based kinetic model for evaluating receptor-mediated endocytotic processes in which receptors recycle.


The process of receptor-mediated endocytosis for receptors that recycle to the cell surface in an active form can be considered as being kinetically analogous to that of a uni-substrate, uni-product enzyme-catalysed reaction. In this study we have derived steady-state initial-velocity rate equations for this process, based on classical Briggs-Haldane and King-Altman kinetic approaches to multi-step reactions, and have evaluated this kinetic paradigm, using as a model system the low-density lipoprotein (LDL)-receptor-mediated endocytosis of the trapped label [14C]sucrose-LDL in uninduced, steady-state Hep-G2 cells. Using the derived rate equations, together with experimentally determined values for Bmax (123 fmol/mg of cell protein), Kd (14.3 nM), the endocytotic rate constant ke (analogous to kcat; 0.163 min-1), Km (80 nM) and maximal internalization velocity (26.4 fmol/min per mg), we have calculated the ratio ke/Km (0.00204 nM-1.min-1), the bimolecular rate constant for LDL and LDL-receptor association (0. 00248 nM-1.min-1), the first-order rate constant for LDL-LDL-receptor complex dissociation (0.0354 min-1), the total cellular content of LDL receptors (154 fmol/mg of cell protein), the intracellular LDL receptor concentration (30.7 fmol/mg of cell protein) and the pseudo-first-order rate constant for LDL receptor recycling (0.0653 min-1). Based on this mathematical model, the kinetic mechanism for the receptor-mediated endocytosis of [14C]sucrose-LDL by steady-state Hep-G2 cells is one of constitutive endocytosis via independent internalization sites that follows steady-state Briggs-Haldane kinetics, such that LDL-LDL-receptor interactions are characterized by a rapid-high-affinity ligand-receptor association, followed by ligand-receptor complex internalization that is rapid relative to complex dissociation, and by receptor recycling that is more rapid than complex internalization and that serves to maintain 80% of cellular LDL receptors on the cell surface in the steady-state. The consistency with which these quantitative observations parallel previous qualitative observations regarding LDL-receptor-mediated endocytosis, together with the high correlation between theoretical internalization velocities (calculated from determined rate constants) and experimental internalization velocities, underscore the validity of considering receptor-mediated endocytotic processes for recycling receptors in catalytic terms.

Full Text

The Full Text of this article is available as a PDF (522K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986 Apr 4;232(4746):34–47. [PubMed]
  • Brown MS, Anderson RG, Goldstein JL. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. [PubMed]
  • Pastan IH, Willingham MC. Journey to the center of the cell: role of the receptosome. Science. 1981 Oct 30;214(4520):504–509. [PubMed]
  • Goldstein JL, Anderson RG, Brown MS. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. [PubMed]
  • Goldstein JL, Brown MS, Anderson RG, Russell DW, Schneider WJ. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. [PubMed]
  • Goldstein JL, Brown MS. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. [PubMed]
  • Kaplan J. Polypeptide-binding membrane receptors: analysis and classification. Science. 1981 Apr 3;212(4490):14–20. [PubMed]
  • Wiley HS, Cunningham DD. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed]
  • Wiley HS, Cunningham DD. A steady state model for analyzing the cellular binding, internalization and degradation of polypeptide ligands. Cell. 1981 Aug;25(2):433–440. [PubMed]
  • Lund KA, Opresko LK, Starbuck C, Walsh BJ, Wiley HS. Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization. J Biol Chem. 1990 Sep 15;265(26):15713–15723. [PubMed]
  • Knauer DJ, Wiley HS, Cunningham DD. Relationship between epidermal growth factor receptor occupancy and mitogenic response. Quantitative analysis using a steady state model system. J Biol Chem. 1984 May 10;259(9):5623–5631. [PubMed]
  • Wiley HS. Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system. J Cell Biol. 1988 Aug;107(2):801–810. [PMC free article] [PubMed]
  • Pitas RE, Innerarity TL, Arnold KS, Mahley RW. Rate and equilibrium constants for binding of apo-E HDLc (a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: evidence for multiple receptor binding of apo-E HDLc. Proc Natl Acad Sci U S A. 1979 May;76(5):2311–2315. [PMC free article] [PubMed]
  • Pedreño J, de Castellarnau C, Cullaré C, Sánchez J, Gómez-Gerique J, Ordóez-Llanos J, González-Sastre F. LDL binding sites on platelets differ from the "classical" receptor of nucleated cells. Arterioscler Thromb. 1992 Nov;12(11):1353–1362. [PubMed]
  • Javitt NB. Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids. FASEB J. 1990 Feb 1;4(2):161–168. [PubMed]
  • Opresko LK, Wiley HS. Receptor-mediated endocytosis in Xenopus oocytes. I. Characterization of the vitellogenin receptor system. J Biol Chem. 1987 Mar 25;262(9):4109–4115. [PubMed]
  • Opresko LK, Wiley HS. Receptor-mediated endocytosis in Xenopus oocytes. II. Evidence for two novel mechanisms of hormonal regulation. J Biol Chem. 1987 Mar 25;262(9):4116–4123. [PubMed]
  • Johnson CL, Johnson CG. Characterization of receptors for substance P in human astrocytoma cells: radioligand binding and inositol phosphate formation. J Neurochem. 1992 Feb;58(2):471–477. [PubMed]
  • Servant G, Boulay G, Bossé R, Escher E, Guillemette G. Photoaffinity labeling of subtype 2 angiotensin receptor of human myometrium. Mol Pharmacol. 1993 May;43(5):677–683. [PubMed]
  • Havekes L, van Hinsbergh V, Kempen HJ, Emeis J. The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2. Biochem J. 1983 Sep 15;214(3):951–958. [PMC free article] [PubMed]
  • Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. [PubMed]
  • Rajan VP, Menon KM. Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells. Endocrinology. 1985 Dec;117(6):2408–2416. [PubMed]
  • Pittman RC, Taylor CA., Jr Methods for assessment of tissue sites of lipoprotein degradation. Methods Enzymol. 1986;129:612–628. [PubMed]
  • Goldstein JL, Brown MS. Binding and degradation of low density lipoproteins by cultured human fibroblasts. Comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia. J Biol Chem. 1974 Aug 25;249(16):5153–5162. [PubMed]
  • Dashti N, Wolfbauer G, Koren E, Knowles B, Alaupovic P. Catabolism of human low density lipoproteins by human hepatoma cell line HepG2. Biochim Biophys Acta. 1984 Jul 26;794(3):373–384. [PubMed]
  • Pittman RC, Carew TE, Attie AD, Witztum JL, Watanabe Y, Steinberg D. Receptor-dependent and receptor-independent degradation of low density lipoprotein in normal rabbits and in receptor-deficient mutant rabbits. J Biol Chem. 1982 Jul 25;257(14):7994–8000. [PubMed]
  • Pittman RC, Steinberg D. A new approach for assessing cumulative lysosomal degradation of proteins or other macromolecules. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1254–1259. [PubMed]
  • Pittman RC, Green SR, Attie AD, Steinberg D. Radiolabeled sucrose covalently linked to protein. A device for quantifying degradation of plasma proteins catabolized by lysosomal mechanisms. J Biol Chem. 1979 Aug 10;254(15):6876–6879. [PubMed]
  • Larkin JM, Donzell WC, Anderson RG. Modulation of intracellular potassium and ATP: effects on coated pit function in fibroblasts and hepatocytes. J Cell Physiol. 1985 Sep;124(3):372–378. [PubMed]
  • Anderson RG, Brown MS, Goldstein JL. Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells. J Cell Biol. 1981 Feb;88(2):441–452. [PMC free article] [PubMed]
  • Green SA, Kelly RB. Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells. J Cell Biol. 1992 Apr;117(1):47–55. [PMC free article] [PubMed]
  • Bos CR, Shank SL, Snider MD. Role of clathrin-coated vesicles in glycoprotein transport from the cell surface to the Golgi complex. J Biol Chem. 1995 Jan 13;270(2):665–671. [PubMed]
  • Spurlock ME, Cusumano JC, Mills SE. (-)-[3H]-dihydroalprenolol binding to beta-adrenergic receptors in porcine adipose tissue and skeletal muscle membrane preparations. J Anim Sci. 1993 Jul;71(7):1778–1785. [PubMed]
  • Baron BM, Siegel BW. p-[125I]iodoclonidine, a novel radiolabeled agonist for studying central alpha 2-adrenergic receptors. Mol Pharmacol. 1990 Sep;38(3):348–356. [PubMed]
  • Feifel R, Rodrigues de Miranda JF, Strohmann C, Tacke R, Aasen AJ, Mutschler E, Lambrecht G. Selective labelling of muscarinic M1 receptors in calf superior cervical ganglia by [3H](+/-)-telenzepine. Eur J Pharmacol. 1991 Mar 19;195(1):115–123. [PubMed]
  • Garlind A, Cowburn RF, Fowler CJ. Characterization of [3H]inositol 1,4,5-trisphosphate binding sites in human temporal cortical and cerebellar membranes. Neurochem Int. 1994 Jan;24(1):73–80. [PubMed]
  • Casadó V, Mallol J, Lluis C, Canela EI, Franco R. Effect of phospholipases and proteases on the [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) binding to A1 adenosine receptors from pig cerebral cortex. J Cell Biochem. 1991 Nov;47(3):278–288. [PubMed]
  • Furfine ES, Leban JJ, Landavazo A, Moomaw JF, Casey PJ. Protein farnesyltransferase: kinetics of farnesyl pyrophosphate binding and product release. Biochemistry. 1995 May 23;34(20):6857–6862. [PubMed]
  • Adams JA, Taylor SS. Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments. Biochemistry. 1992 Sep 15;31(36):8516–8522. [PubMed]
  • Otlewski J, Zbyryt T. Single peptide bond hydrolysis/resynthesis in squash inhibitors of serine proteinases. 1. Kinetics and thermodynamics of the interaction between squash inhibitors and bovine beta-trypsin. Biochemistry. 1994 Jan 11;33(1):200–207. [PubMed]
  • Stivers JT, Shuman S, Mildvan AS. Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions. Biochemistry. 1994 Jan 11;33(1):327–339. [PubMed]
  • Beebe JA, Fierke CA. A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P. Biochemistry. 1994 Aug 30;33(34):10294–10304. [PubMed]
  • Ikebe M, Hartshorne DJ. Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP. J Biol Chem. 1986 Jun 25;261(18):8249–8253. [PubMed]
  • Shacter E, Chock PB, Stadtman ER. Regulation through phosphorylation/dephosphorylation cascade systems. J Biol Chem. 1984 Oct 10;259(19):12252–12259. [PubMed]
  • Simon DI, Ezratty AM, Loscalzo J. The fibrin(ogen)olytic properties of cathepsin D. Biochemistry. 1994 May 31;33(21):6555–6563. [PubMed]
  • Stack MS, Pizzo SV. The effect of substituted laminin A chain-derived peptides on the conformation and activation kinetics of plasminogen. Arch Biochem Biophys. 1994 Feb 15;309(1):117–122. [PubMed]
  • Maslak M, Martin CT. Effects of solution conditions on the steady-state kinetics of initiation of transcription by T7 RNA polymerase. Biochemistry. 1994 Jun 7;33(22):6918–6924. [PubMed]
  • Warwicker J, Mueller-Harvey I, Sumner I, Bhat KM. The activity of porcine pancreatic phospholipase A2 in 20% alcohol/aqueous solvent, by experiment and electrostatics calculations. J Mol Biol. 1994 Feb 25;236(3):904–917. [PubMed]
  • Zhang D, Jennings SM, Robinson GW, Poulter CD. Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Arch Biochem Biophys. 1993 Jul;304(1):133–143. [PubMed]
  • Song L, Poulter CD. Yeast farnesyl-diphosphate synthase: site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3044–3048. [PMC free article] [PubMed]
  • Yokoyama K, McGeady P, Gelb MH. Mammalian protein geranylgeranyltransferase-I: substrate specificity, kinetic mechanism, metal requirements, and affinity labeling. Biochemistry. 1995 Jan 31;34(4):1344–1354. [PubMed]
  • Pompliano DL, Rands E, Schaber MD, Mosser SD, Anthony NJ, Gibbs JB. Steady-state kinetic mechanism of Ras farnesyl:protein transferase. Biochemistry. 1992 Apr 21;31(15):3800–3807. [PubMed]

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


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...