Abstract
Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.
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- Alvarado F. Hypothesis for the interaction of phlorizin and phloretin with membrane carriers for sugars. Biochim Biophys Acta. 1967 Jul 3;135(3):483–495. doi: 10.1016/0005-2736(67)90038-7. [DOI] [PubMed] [Google Scholar]
- Benes I., Kolínská J., Kotyk A. Effect of phloretin on monosaccharide transport in erythrocyte ghosts. J Membr Biol. 1972;8(3):303–309. doi: 10.1007/BF01868107. [DOI] [PubMed] [Google Scholar]
- Bihler I., Cavert H. M., Fisher R. H. A differential effect of inhibitors on sugar penetration into the isolated rabbit heart. J Physiol. 1965 Sep;180(1):168–177. [PMC free article] [PubMed] [Google Scholar]
- Brown P. A., Feinstein M. B., Sha'afi R. I. Membrane proteins related to water transport in human erythrocytes. Nature. 1975 Apr 10;254(5500):523–525. doi: 10.1038/254523a0. [DOI] [PubMed] [Google Scholar]
- Burger S. P., Fujii T., Hanahan D. J. Stability of the bovine erythrocyte membrane. Release of enzymes and lipid components. Biochemistry. 1968 Oct;7(10):3682–3700. doi: 10.1021/bi00850a048. [DOI] [PubMed] [Google Scholar]
- CHAN S. S., LOTSPEICH W. D. Comparative effects of phlorizin and phloretin on glucose transport in the cat kidney. Am J Physiol. 1962 Dec;203:975–979. doi: 10.1152/ajplegacy.1962.203.6.975. [DOI] [PubMed] [Google Scholar]
- Cabantchik Z. I., Rothstein A. Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J Membr Biol. 1974;15(3):207–226. doi: 10.1007/BF01870088. [DOI] [PubMed] [Google Scholar]
- Cass A., Finkelstein A. Water permeability of thin lipid membranes. J Gen Physiol. 1967 Jul;50(6):1765–1784. doi: 10.1085/jgp.50.6.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Czech M. P., Lynn D. G., Lynn W. S. Cytochalasin B-sensitive 2-deoxy-D-glucose transport in adipose cell ghosts. J Biol Chem. 1973 May 25;248(10):3636–3641. [PubMed] [Google Scholar]
- Czech M. P., Lynn D. G., Lynn W. S. Phlorizin--receptor interactions in fat cell plasma membranes. Biochim Biophys Acta. 1973 Nov 16;323(4):639–642. doi: 10.1016/0005-2736(73)90175-2. [DOI] [PubMed] [Google Scholar]
- DITTRICH H. DIE MITTLERE HAEMOGLOBIN-KONZENTRATION DER ERYTHROZYTEN. Med Klin. 1963 Nov 15;58:1882–1884. [PubMed] [Google Scholar]
- DODGE J. T., MITCHELL C., HANAHAN D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119–130. doi: 10.1016/0003-9861(63)90042-0. [DOI] [PubMed] [Google Scholar]
- Dalmark M., Wieth J. O. Temperature dependence of chloride, bromide, iodide, thiocyanate and salicylate transport in human red cells. J Physiol. 1972 Aug;224(3):583–610. doi: 10.1113/jphysiol.1972.sp009914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Diedrich D. F. Is phloretin the sugar transport inhibitor in intestine? Arch Biochem Biophys. 1968 Sep 20;127(1):803–812. doi: 10.1016/0003-9861(68)90292-0. [DOI] [PubMed] [Google Scholar]
- ELLMAN G. L., COURTNEY K. D., ANDRES V., Jr, FEATHER-STONE R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88–95. doi: 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
- Everitt C. T., Redwood W. R., Haydon D. A. Problem of boundary layers in the exchange diffusion of water across bimolecular lipid membranes. J Theor Biol. 1969 Jan;22(1):20–32. doi: 10.1016/0022-5193(69)90077-0. [DOI] [PubMed] [Google Scholar]
- Glossmann H., Neville D. M., Jr Phlorizin receptors in isolated kidney brush border membranes. J Biol Chem. 1972 Dec 10;247(23):7779–7789. [PubMed] [Google Scholar]
- Guidotti G. The composition of biological membranes. Arch Intern Med. 1972 Feb;129(2):194–201. [PubMed] [Google Scholar]
- HUMMEL J. P., DREYER W. J. Measurement of protein-binding phenomena by gel filtration. Biochim Biophys Acta. 1962 Oct 8;63:530–532. doi: 10.1016/0006-3002(62)90124-5. [DOI] [PubMed] [Google Scholar]
- Kahlenberg A., Urman B., Dolansky D. Preferential uptake of D-glucose by isolated human erythrocyte membranes. Biochemistry. 1971 Aug 3;10(16):3154–3162. doi: 10.1021/bi00792a027. [DOI] [PubMed] [Google Scholar]
- Kaplan M. A., Hays L., Hays R. M. Evolution of a facilitated diffusion pathway for amides in the erythrocyte. Am J Physiol. 1974 Jun;226(6):1327–1332. doi: 10.1152/ajplegacy.1974.226.6.1327. [DOI] [PubMed] [Google Scholar]
- Krupka R. M. Evidence for a carrier conformational change associated with sugar transport in erythrocytes. Biochemistry. 1971 Mar 30;10(7):1143–1148. doi: 10.1021/bi00783a007. [DOI] [PubMed] [Google Scholar]
- LEFEVRE P. G., MARSHALL J. K. The atachment of phloretin and analogues to human erythrocytes in connection with inhibition of sugar transport. J Biol Chem. 1959 Nov;234:3022–3026. [PubMed] [Google Scholar]
- LEFEVRE P. G. Sugar transport in the red blood cell: structure-activity relationships in substrates and antagonists. Pharmacol Rev. 1961 Mar;13:39–70. [PubMed] [Google Scholar]
- Levine S., Franki N., Hays R. M. Effect of phloretin on water and solute movement in the toad bladder. J Clin Invest. 1973 Jun;52(6):1435–1442. doi: 10.1172/JCI107317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin S., Spudich J. A. Biochemical studies on the mode of action of cytochalasin B. Cytochalasin B binding to red cell membrane in relation to glucose transport. J Biol Chem. 1974 Sep 25;249(18):5778–5783. [PubMed] [Google Scholar]
- Macey R. I., Farmer R. E. Inhibition of water and solute permeability in human red cells. Biochim Biophys Acta. 1970 Jul 7;211(1):104–106. doi: 10.1016/0005-2736(70)90130-6. [DOI] [PubMed] [Google Scholar]
- Owen J. D., Solomon A. K. Control of nonelectrolyte permeability in red cells. Biochim Biophys Acta. 1972 Dec 1;290(1):414–418. doi: 10.1016/0005-2736(72)90087-9. [DOI] [PubMed] [Google Scholar]
- Owen J. D., Steggall M., Eyring E. M. The effect of phloretin on red cell nonelectrolyte permeability. J Membr Biol. 1974;19(1):79–92. doi: 10.1007/BF01869971. [DOI] [PubMed] [Google Scholar]
- Poznansky M., Tong S., White P. C., Milgram J. M., Solomon A. K. Nonelectrolyte diffusion across lipid bilayer systems. J Gen Physiol. 1976 Jan;67(1):45–66. doi: 10.1085/jgp.67.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- SINGLETON W. S., GRAY M. S., BROWN M. L., WHITE J. L. CHROMATOGRAPHICALLY HOMOGENEOUS LECITHIN FROM EGG PHOSPHOLIPIDS. J Am Oil Chem Soc. 1965 Jan;42:53–56. doi: 10.1007/BF02558256. [DOI] [PubMed] [Google Scholar]
- Seeman P., Roth S. General anesthetics expand cell membranes at surgical concentrations. Biochim Biophys Acta. 1972 Jan 17;255(1):171–177. doi: 10.1016/0005-2736(72)90019-3. [DOI] [PubMed] [Google Scholar]
- Seeman P. The membrane actions of anesthetics and tranquilizers. Pharmacol Rev. 1972 Dec;24(4):583–655. [PubMed] [Google Scholar]
- Taverna R. D., Langdon R. G. A new method for measuring glucose translocation through biological membranes and its application to human erythrocyte ghosts. Biochim Biophys Acta. 1973 Mar 16;298(2):412–421. doi: 10.1016/0005-2736(73)90368-4. [DOI] [PubMed] [Google Scholar]
- Taverna R. D., Langdon R. G. Reversible association of cytochalasin B with the human erythrocyte membrane. Inhibition of glucose transport and the stoichiometry of cytochalasin binding. Biochim Biophys Acta. 1973 Oct 11;323(2):207–219. doi: 10.1016/0005-2736(73)90145-4. [DOI] [PubMed] [Google Scholar]