Proc. Nat. Acad. Sci. USA Vol. 73, No. 2, pp. 396-400, February 1976
Biochemistry
Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes (monosaccharide transport/membrane protein/Triton X-100/octylglucoside)
MICHIHIRo KASAHARA AND PETER C. HINKLE Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
Communicated by Leon A. Heppel, November 21, 1975
lution and twice with 50 ml of 10 mM Tris-H2SO4 (pH 7.4), and stored at -70° (5 mg of protein per ml in 10 mM Tris, H2SO4). We confirmed the previous observation that EDTA and NaCl treatment removed several proteins from the ghosts and converted the ghosts into small vesicles (25). Solubilization with detergents Solubilization with Triton X-100. Triton X-100 was treated with SnCl2 to remove peroxides (26, 27). Most of the peroxides (more than 85%) were removed, as estimated by the ferrous thiocyanate method (28). Sodium-chloride-treated vesicles (40 mg of protein in 20 ml) were incubated with 0.5% of purified Triton X-100 in 10 mM Tris-H2SO4 (pH 7.4) for 20 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was treated with Bio-Beads SM-2 overnight (6 g wet weight of beads per 20 ml) at 40 (29), divided into small fractions, and stored at -70°. Solubilization with Octylglucoside. Sodium-chloridetreated vesicles (14 mg of protein in 7 ml) were incubated with 30 mM octylglucoside in 10 mM Tris-H2SO4 (pH 7.4) for 30 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was subjected to diafiltration (XM-50; cut-off molecular weight, 50,000) against 10 mM TrisH2SO4 (pH 7.4) for 6 hr to overnight at 40. The protein fraction was divided into small fractions and stored at -70°. Reconstitution of D-glucose uptake system Acetone-washed soybean phospholipids (16) in a deoxygenated buffer (150 mM KC1, 10 mM Tris.HCl, pH 7.4) were sonicated in a test tube for 15-30 min at 20-400 with a sonicator (80 W, 80 kHz, Generator model G80-80-1, Tank model T80-80-1-R$, Laboratory Supplies, Hicksville, N.Y.) at 25-40 mg dry weight of lipid per ml. The solubilized membrane fraction was added to the sonicated liposomes, 15-20 mg/ml of phospholipids and 0.5-1.0 mg/ml of protein (total volume, 0.5-1.5 ml). The mixture was flushed with N2 gas and sonicated further for 1-3 min at 30-35' (30). The reconstitution was not specific for the soybean phospholipids; a mixture of egg phosphatidylcholine and soybean phosphatidylethanolamine could be used in place of the soybean phospholipids. Uptake of radioactive sugars A portion (85-120 gl) of the reconstituted liposomes was mixed with a buffer (final concentration, 150 mM KCl, 2 mM MgSO4, 10 mM Tris-HCl, pH 7.4; total volume, 200 ,ul, temperature 22-260) and uptake was started by addition of radioactive sugars (0.4 ,uCi of D-[14C]glucose or L-[14C]glucose, final concentration, 0.2 mM). The reaction was stopped with a cold stopping solution (1 mM HgCl2, 150 mM KC1, 2 mM MgSO4, 10 mM Tris-HCl, pH 7.4, temperature 40).
ABSTRACT A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The uptake was time dependent and inhibited by mercuric ions or cytochalasin B. The results indicate that the uptake represents transport of the sugar into the liposomes ratier thanbinding to the reconstituted liposomes.
The transport system for monosaccharides in human erythrocytes is one of the most extensively studied facilitated diffusion systems (1-3). Although kinetic analyses have been performed in detail (4, 5), attempts to isolate the component(s) involved have begun only recently. In inhibitor binding studies, band 3 protein (6), bands 3 and 4 (7), or another protein (molecular weight 180,000) (8) were implicated in this sugar transport [nomenclature is according to Steck (9)]. Other attempts to isolate the essential component(s) of the sugar transport system based on glucose binding or reconstitution were not successful (2, 10-14, see also ref. 15). However, other membrane transport systems have been reconstituted in liposomes by several methods (16-22). In this report, we show that a solubilized membrane protein fraction from human erythrocytes containing band 3 protein and other minor proteins is capable of D-glucose transport when reconstituted into liposomes by sonication. MATERIALS AND METHODS Materials D_[14C]Glucose and L-[14C]glucose were purchased from New England Nuclear; Triton X-100, Tween 80, and soybean lipid (L-a-phosphatidylcholine, Type II-S), from Sigma; cytochalasin B, from Imperial Chemical; Bio-Beads SM-2, from Bio-Rad; membrane filters, from Millipore and Schleicher and Schuell; ultrafiltration membrane XM-50, from Amicon; and Sephadex G-50 medium, from Pharmacia. Octylglucoside (23) was a gift from Dr. E. Racker, this department. Preparation of the membrane fraction Ghosts were prepared from newly outdated human erythrocytes according to Dodge et al. (24) and stored at -70°. Ghosts were treated with EDTA and NaCl according to the procedure of Fairbanks et al. (25) with a slight modification. Ghosts (150 mg of protein in 30 ml) were thawed, treated with 10 volumes of 0.1 mM EDTA (pH 8.0) for 20 min at 370, and sedimented at 78,000 X g for 30 min. The pellet was washed once with 100 ml of 0.1 mM EDTA, treated with 100 ml of 0.5 M NaCl, 5 mM Tris-H2SO4 (pH 7.4) for 20 min at 40, washed once with 50 ml of the same salt so396
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Kasahara and Hinkle
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40
60
80
100
120
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FIG. 2. Time course of D-glucose uptake. The reconstituted li(0.7 mg/ml of protein solubilized with Triton X-100 and 15 mg/ml of phospholipids) were prepared as described in Materials and Methods. (A) Rapid uptake of D-glucose. Uptake was started by addition of 0.2 mM D- or L-[14C]glucose to the reaction mixture containing reconstituted liposomes (56 gg of protein and 1.2 mg of phospholipids). At indicated times, it was stopped with a cold stopping solution containing mercuric ions and followed by filtration through a membrane filter. (B) Uptake of D- or L-glucose for longer periods. The same assay conditions as in (A) were used. posomes
A
B
C
FIG. 1. Sodium dodecyl sulfate-gel electrophoresis of ghosts (A), Triton-X-100-solubilized protein fraction (B), and octylglucoside-solubilized protein fraction (C). About 50 ,gg of protein was applied to each gel and stained with Coomassie blue. The tracking dye was bromophenol blue (BPB).
Gel Filtration Method. After addition of 0.3 ml of the stopping solution, the uptake medium was applied to a Sephadex column (G-50 medium, 25 cm X 0.7 cm) and elution was performed at 40 with the stopping solution. It took 15-20 min from the end of uptake until the elution of the
liposome fraction. Radioactivity of each fraction (0.6 ml) counted in Bray's solution (31). Membrane Filter Method. After addition of 5 ml of the stopping solution, the uptake medium was passed through a membrane filter (Selectron 0.2 gm or Millipore 0.22 gm), followed by a 10 ml wash with the cold stopping solution. Radioactivity was counted in Bray's solution. When a proper amount of the reconstituted liposomes (less than 2 mg of phospholipids) was used, the membrane filter method showed more than 80% of D-glucose retention observed with the gel filtration method. Analytical methods Protein (32), phosphorus (33), Triton X-100 (29), and octylglucoside (23) were measured as described. Sodium dodecyl sulfate gel electrophoresis (5% acrylamide, 0.2% sodium dodecyl sulfate) was performed according to the published method (25, 34). Each gel was stained with Coomassie blue G, periodic acid-Schiff reagent (25), or a carbocyanine dye (35). Paper chromatography of sugars was performed using Whatman no. 4 paper and a solvent mixture of n-butanol, ethanol, and water (50:32:18). When the D-glucose uptake in the reconstituted liposomes was assayed with the membrane filter method more than 90% of the radioisotope on the filter co-migrated with D-glucose.
was
RESULTS AND DISCUSSION Properties of the solubilized protein fractions *About half of the protein (47%) was removed from ghosts by the EDTA-NaCl treatment. The washed vesicles were incubated with non-ionic detergents (Triton X-100 or octylglucoside). Both detergents solubilized a considerable amount of protein from the vesicles (70% with Triton X-100 and 56% with octylglucoside). The protein fraction solubilized with Triton X-100 contained several proteins, of which a main protein was band 3 (36), and octylglucoside gave the same result (Fig. 1). Four glycoprotein bands (PAS 1-4) were also observed with periodic acid-Schiff staining or a carbocyanine dye staining (not shown). Phospholipids were also solubilized; 0.39 ,mol of total P per mg of protein with Triton X-100 and 0.56 Amol of total P per mg of protein with octylglucoside. Satisfactory removal of detergents was achieved by the Bio-Beads SM-2 treatment or diafiltration. Ultrafiltration was performed to determine the free detergent concentration in the final protein fractions; the ultrafiltrate contained 0.002% Triton X-100 or 10 WM octylglucoside. These values were well below the critical micelle concentrations of the detergents (23, 29). Characteristics of D-glucose uptake The reconstituted liposomes showed time-dependent, rapid D-glucose uptake, with a half time of 6 sec (Fig. 2A). To estimate the nonspecific transport and/or binding, L-glucose, which is not a substrate for the monosaccharide transport system in human erythrocytes (1), was used. The uptake of L-glucose showed a slower time course. The initial rate of uptake was 36 times faster for D-glucose than for L-glucose.
898
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Kasahara and Hinkle
Table 1. Substrate specificity of the reconstituted sugar. uptake system
Uptake (percent of total radioisotope/15 sec) Fraction of*
rapid Addition
D-Glucose
L-Glucose
D-glucose uptake
D-Glucose 2-Deoxy-D-glucose 3-O-Methyl-D-glucose D-Mannose D-Galactose D-Ribose L-Glucose
0.054 0.022 0.027 0.025 0.025 0.027 0.048 0.048
0.012 0.009 0.009 0.009 0.010 0.009 0.010 0.011
0.042 0.013 0.018 0.016 0.015 0.018 0.038 0.037
Percent inhibition 69 57 62 64 57 10 12
Each sugar (20 mM) with 0.2 mM D- or L-[14C]glucose was added to the reaction mixture containing reconstituted vesicles (86,gg of protein solubilized with Triton X-100 and 1.2 mg of phospholipids) to start the uptake. Uptake was measured with the membrane filter method. * Fraction of rapid D-glucose uptake was calculated by subtracting L-glucose uptake from D-glucose uptake.
After the rapid phase, uptake of D-glucose showed a slow phase which continued for about 2 hr (Fig. 2B). Uptake of L-glucose showed only the slow phase with a half time of about 30 min and appeared to approach the same final extent as D-glucose uptake. At zero time, a small apparent uptake of both isomers was observed. Since liposomes without protein showed a slow uptake of both isomers and similar zero time uptake (not shown), the rapid uptake of D-glucose shown in Fig. 2A is the component dependent on the protein fraction. The slow uptake of both isomers in reconstituted Iiposomes may indicate penetration via simple diffusion and the zero time uptake may result from nonspecific retention of isotopes. The extent of the rapid D-glucose uptake (30 see) corresponded to 48% of the total extent (2 hr) in the experiment shown in Fig. 2, but this value showed considerable Table 2. Effect of inhibitors on the reconstituted system
Uptake, pmol/mg of protein Substrate
Addition
per 15 sec
Exp. 1* 113
D-Glucose
L-Glucose
HgCl2, 20,.iM HgCl2, 600 pM HgCl2, 600 PM
D-Glucose
Exp. 2 Ethanol, 1%
Cytochalasin Bt, 1 ,uMt Cytochalasin B, 5 JM L-Glucose
Ethanol, 1%
variation from 18% to 60%, in other experiments. No signifidifference of uptake activity was found between the Triton-X-100- and octylglucoside-solubilized protein fractions.
cant
The substrate specificity of the D-glucose uptake system studied by inhibition with nonradioactive sugars (Table 1). D-Glucose uptake was inhibited by several sugars (D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-mannose, D-galactose) which are known to have affinity for the monosaccharide transport system in erythrocytes (1, 37). L-Glucose and D-ribose did not inhibit well, as expected from the results on the erythrocytes. D-Glucose uptake was inhibited by two reagents. Table 2 shows that mercuric ions and cytochalasin B inhibited the uptake at the concentration comparable to that for erythrocytes (38-40). Stilbestrol, which inhibits monosaccharide transport in erythrocytes and ghosts (1, 41), did not inhibit the reconstituted system even at 50 ,uM. This might indicate that some factors are missing in the present reconstituted
was
system.
The possibility that the results reflect binding instead of ways. The uptake was de-
transport was examined in several
Table 3. Effect of lipid and protein
34 34 25 49
123 82 51 38 52 37
Cytochalasin B, 1 JM Cytochalasin B, 5 ,M The Triton-X-100-solubilized protein fraction was used for the reconstitution. Phospholipid concentration after sonication was 15. mg/ml (Exp. 1) or 20 mg/ml (Exp. 2) and that of protein was 0.75 mg/ml (Exp. 1) or 0.80 mg/ml (Exp. 2). Uptake was measured with the gel filtration method (Exp. 1) or the membrane filter method (Exp. 2). * Uptake was measured at 100. t Cytochalasin B was dissolved in ethanol and mixed in the assay mixture. Maximum ethanol concentration in the assay was 1%.
on
D-glucose uptake
Uptake (percent of total radioisotope)
Reconstituted liposomes* Protein alonet Liposomes alone t
D-Glucose
L-Glucose
0.110 0.004 0.020
0.037 0.026
The uptake was measured with the gel filtration method for 1 min. * The reconstituted liposomes (15 mg/ml of phospholipids and 0.75 mg/ml of protein solubilized with Triton X-100) were prepared as described in Materials and Methods. A portion of 140 gl (0.1 mg of protein and 2.1 mg of phospholipids) was used for each uptake assay. t "Protein alone" was prepared by adjusting the KCI concentration of the protein fraction to 150 mM and 0.1 mg of protein was used for each assay. "Liposomes alone" was prepared in the same way as "Reconstituted liposomes" except that Tris-KCI mixture was added in place of protein and 2.1 mg of phospholipids was used for each assay.
Biochemistry:
Kasahara and Hinkle
004|-\
D-glucose
003 o
L-glucose
0
Proc. Nat. Acad. Sci. USA 73 (1976)
We wish to thank Dr. L. A. Heppel, who was associated with this research, for many discussions and continual encouragement. We are also grateful to Dr. E. Racker for providing octylglucoside and stimulating discussions and criticisms. This study was supported by National Institutes of Health Grant no. CA-14454. 1. LeFevre, P. G. (1961) Pharmacol. Rev. 13, 39-70. 2. Stein, W. D. (1967) in The Movement of Molecules Across Cell Membranes (Academic Press, New York), pp. 126-176 and pp. 266-308.
0.02
C1
0.01 _ o
0
5
10
15
sonication time (minutes)
FIG. 3. Dependence of the reconstitution on sonication time. Liposomes were mixed with the Triton-X-100-solubilized protein fraction (15 mg/ml of phospholipids and 0.59 mg/ml of protein) and sonicated for the indicated time at 350. A portion of 120 ,u was used for a 15 sec uptake assay by the membrane filter method.
pendent
399
lipid
on both and was essential for
the protein fraction (Table 3). the reconstitution (Fig. 3), but sonication times longer than 3 min were inhibitory. The liposomes showed loss of D-['4C]glucose when chased with nonradioactive D-glucose (Fig. 4). The final extent of D['4C]glucose uptake in the liposomes returned almost to the original level after this transient loss. Triton X-100 (0.5%) or Tween 80 (0.5%) inhibited the D-glucose uptake (30 sec) by 89% and 81%, respectively (not shown). At a higher concentration of D-glucose (20 mM), the initial rate of uptake was 4.5 nmol/mg of protein per 10 sec and the extent of D-glucose taken up reached 14 nmol/mg of protein at 5 min (not shown). This extent is 3.5 times higher than the upper limit of binding capacity in the protein fraction if we assume that one protein molecule binds maximally one sugar molecule, and that 40% of the protein fraction, which corresponds to the maximum fraction of a single protein (band 3), is the glucose binding protein and the molecular weight of the protein is 100,000. These results are consistent with the idea that the uptake of D-glucose is transport of the sugar into the reconstituted liposomes via a specific transport system.. The isolation of a transport system and reconstitution into a well-defined membrane are indispensable for the study of transport at the molecular level. The present work is a first step toward such studies of the monosaccharide transport system of human erythrocytes.
Sonication
FIG. 4. Transient loss of accumulated radioactive D-glucose induced by addition of nonradioactive D-glucose. At 30 sec, nonradioactive 10 mM D- or L-glucose was added to the assay mixture (closed symbols). The assay mixture contained reconstituted liposomes (56 /g of protein solubilized with Triton X-100 and 1.2 mg of phospholipids). Uptake was measured by the membrane filter method.
3. Kotyk, A. (1975) in Cell Membrane Transport. Principles and Techniques, eds. Kotyk, A. & Janfacek, K. (Plenum Press, New York), pp. 391-395. 4. Lieb, W. R. & Stein, W. D. (1972) Biochim. Biophys. Acta 265, 187-207. 5. LeFevre, P. G. (1973) J. Membr. Biol. 11, 1-19. 6. Lin, S. & Spudich, J. A. (1974) Biochem. Biophys. Res. Commun. 61, 1471-1476. 7. Taverna, R. D. & Langdon, R. G. (1973) Biochem. Biophys. Res. Commun. 54,593-599. 8. Jung, C. Y. & Carlson, L. M. (1975) J. Biol. Chem. 250, 3217-3220. 9. Steck, T. L. (1974) J. Cell Biol. 62, 1-19. 10. Wood, R. E., Wirth, F. P., Jr. & Morgan, H. E. (1968) Biochim. Biophys. Acta 163, 171-178. 11. Jung, C. Y., Chaney, J. E. & LeFevre, P. G. (1968) Arch. Biochem. Biophys. 126,664-676. 12. LeFevre, P. G., Jung, C. Y. & Chaney, J. E. (1968) Arch. Biochem. Biophys. 126,677-691. 13. Jung, C. Y. (1971) J. Membr. Biol. 5,200-214. 14. Lidgard, G. P. & Jones, M. N. (1975) J. Membr. Biol. 21, 1-10.
15. LeFevre, P. G. & Masiak, S. J. (1970) J. Membr. Biol. 3,387399. 16. Kagawa, Y. & Racker, E. (1971) J. Biol. Chem. 246, 54775487. 17. Hinkle, P. C., Kim, J. J. & Racker, E. (1972) J. Biol. Chem. 247, 1338-1339. 18. Racker, E. (1972) J. Biol. Chem. 247, 8198-8200. 19. Racker, E. & Stoeckenius, W. (1974) J. Biol. Chem. 249, 662-663. 20. Goldin, S. M. & Tong, S. W. (1974) J. Biol. Chem. 249, 5907-5915. 21. Sb-rtzer, H. G. & Racker, E. (1974) J. Biol. Chem. 249, 1320-1321. 22. Rothstein, A., Cabantchik, Z. I., Balshin, M. & Juliano, R. (1975) Biochem. Biophys. Res. Commun. 64, 144-150. 23. Baron, C. & Thompson, T. E. (1975) Biochim. Biophys. Acta 382,276-285. 24. Dodge, J. T., Mitchell, C. & Hanahan, D. J. (1963) Arch. Biochem. Biophys. 100, 119-130. 25. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617. 26. Privett, 0. S., Lundberg, W. O., Khan, N. A., Tolberg, W. E. & Wheeler, D. H. (1953) J. Am. Oil Chem. Soc. 30,61-66. 27. Witheiler, A. P. (1971) Doctoral Dissertation, Cornell University. 28. Wagner, C. D., Clever, H. L. & Peters, E. D. (as described in) Johnson, R. M. & Siddiqi, I. W. (1970) The Determination of Organic Peroxides (Pergamon Press, Oxford), p. 46. 29. Holloway, P. W. (1973) Anal. Biochem. 53,304-308. 30. Racker, E. (1973) Biochem. Biophys. Res. Commun. 55, 224-230. 31. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 32. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 33. Ames, B. N. & Dubin, D. T. (1960) J. Biol. Chem. 235, 769775. 34. Steck, T. L. & Yu, J. (1973) J. Supramol. Struct. 1, 220-232. 35. Green, M. R. & Pastewka, J. V. (1975) Anal. Biochem. 65, 66-72.
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36. Yu, J., Fischman, D. A. & Steck, T. L. (1973) J. Supramol. Struct. 1,233-248. 37. Barnett, J. E. G., Holman, G. D. & Munday, K. A. (1973) Biochem. J. 131,211-221. 38. LeFevre, P. G. (1948) J. Gen. Physiol. 31, 505-527.
Proc. Nat. Acad. Sci. USA 73 (1976) 39. Bloch, R. (1973) Biochemistry 12,4799-4801. 40. Taverna, R. D. & Langdon, R. G. (1973) Biochim. Biophys. Acta 323,207-219. 41. Jung, C. Y., Carlson, L. M. & Whaley, D. A. (1971) Biochim. Biophys. Acta 241, 613-627.
Biochemistry
Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes (monosaccharide transport/membrane protein/Triton X-100/octylglucoside)
MICHIHIRo KASAHARA AND PETER C. HINKLE Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
Communicated by Leon A. Heppel, November 21, 1975
lution and twice with 50 ml of 10 mM Tris-H2SO4 (pH 7.4), and stored at -70° (5 mg of protein per ml in 10 mM Tris, H2SO4). We confirmed the previous observation that EDTA and NaCl treatment removed several proteins from the ghosts and converted the ghosts into small vesicles (25). Solubilization with detergents Solubilization with Triton X-100. Triton X-100 was treated with SnCl2 to remove peroxides (26, 27). Most of the peroxides (more than 85%) were removed, as estimated by the ferrous thiocyanate method (28). Sodium-chloride-treated vesicles (40 mg of protein in 20 ml) were incubated with 0.5% of purified Triton X-100 in 10 mM Tris-H2SO4 (pH 7.4) for 20 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was treated with Bio-Beads SM-2 overnight (6 g wet weight of beads per 20 ml) at 40 (29), divided into small fractions, and stored at -70°. Solubilization with Octylglucoside. Sodium-chloridetreated vesicles (14 mg of protein in 7 ml) were incubated with 30 mM octylglucoside in 10 mM Tris-H2SO4 (pH 7.4) for 30 min at 4°. After centrifugation at 100,000 X g for 60 min, the supernatant was subjected to diafiltration (XM-50; cut-off molecular weight, 50,000) against 10 mM TrisH2SO4 (pH 7.4) for 6 hr to overnight at 40. The protein fraction was divided into small fractions and stored at -70°. Reconstitution of D-glucose uptake system Acetone-washed soybean phospholipids (16) in a deoxygenated buffer (150 mM KC1, 10 mM Tris.HCl, pH 7.4) were sonicated in a test tube for 15-30 min at 20-400 with a sonicator (80 W, 80 kHz, Generator model G80-80-1, Tank model T80-80-1-R$, Laboratory Supplies, Hicksville, N.Y.) at 25-40 mg dry weight of lipid per ml. The solubilized membrane fraction was added to the sonicated liposomes, 15-20 mg/ml of phospholipids and 0.5-1.0 mg/ml of protein (total volume, 0.5-1.5 ml). The mixture was flushed with N2 gas and sonicated further for 1-3 min at 30-35' (30). The reconstitution was not specific for the soybean phospholipids; a mixture of egg phosphatidylcholine and soybean phosphatidylethanolamine could be used in place of the soybean phospholipids. Uptake of radioactive sugars A portion (85-120 gl) of the reconstituted liposomes was mixed with a buffer (final concentration, 150 mM KCl, 2 mM MgSO4, 10 mM Tris-HCl, pH 7.4; total volume, 200 ,ul, temperature 22-260) and uptake was started by addition of radioactive sugars (0.4 ,uCi of D-[14C]glucose or L-[14C]glucose, final concentration, 0.2 mM). The reaction was stopped with a cold stopping solution (1 mM HgCl2, 150 mM KC1, 2 mM MgSO4, 10 mM Tris-HCl, pH 7.4, temperature 40).
ABSTRACT A protein fraction was obtained from human erythrocyte ghosts by solubilization with Triton X-100 or octylglucoside. Triton X-100 was removed from the protein by Bio-Beads SM-2 and octylglucoside, by diafiltration. The solubilized protein fraction catalyzed D-glucose uptake when reconstituted in sonicated liposomes. The uptake was time dependent and inhibited by mercuric ions or cytochalasin B. The results indicate that the uptake represents transport of the sugar into the liposomes ratier thanbinding to the reconstituted liposomes.
The transport system for monosaccharides in human erythrocytes is one of the most extensively studied facilitated diffusion systems (1-3). Although kinetic analyses have been performed in detail (4, 5), attempts to isolate the component(s) involved have begun only recently. In inhibitor binding studies, band 3 protein (6), bands 3 and 4 (7), or another protein (molecular weight 180,000) (8) were implicated in this sugar transport [nomenclature is according to Steck (9)]. Other attempts to isolate the essential component(s) of the sugar transport system based on glucose binding or reconstitution were not successful (2, 10-14, see also ref. 15). However, other membrane transport systems have been reconstituted in liposomes by several methods (16-22). In this report, we show that a solubilized membrane protein fraction from human erythrocytes containing band 3 protein and other minor proteins is capable of D-glucose transport when reconstituted into liposomes by sonication. MATERIALS AND METHODS Materials D_[14C]Glucose and L-[14C]glucose were purchased from New England Nuclear; Triton X-100, Tween 80, and soybean lipid (L-a-phosphatidylcholine, Type II-S), from Sigma; cytochalasin B, from Imperial Chemical; Bio-Beads SM-2, from Bio-Rad; membrane filters, from Millipore and Schleicher and Schuell; ultrafiltration membrane XM-50, from Amicon; and Sephadex G-50 medium, from Pharmacia. Octylglucoside (23) was a gift from Dr. E. Racker, this department. Preparation of the membrane fraction Ghosts were prepared from newly outdated human erythrocytes according to Dodge et al. (24) and stored at -70°. Ghosts were treated with EDTA and NaCl according to the procedure of Fairbanks et al. (25) with a slight modification. Ghosts (150 mg of protein in 30 ml) were thawed, treated with 10 volumes of 0.1 mM EDTA (pH 8.0) for 20 min at 370, and sedimented at 78,000 X g for 30 min. The pellet was washed once with 100 ml of 0.1 mM EDTA, treated with 100 ml of 0.5 M NaCl, 5 mM Tris-H2SO4 (pH 7.4) for 20 min at 40, washed once with 50 ml of the same salt so396
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Kasahara and Hinkle
sob
D-gIUCOS
A
,c E
500 -a°
E
40
0
30c
V~~~~~~~~~~~~~~
~aE 4400- a/
-
MX
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n
I-
to 0 10 o
~~~oo L. ~~~~L-glucose .2 200
34.14..
397
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20
0
40 time (seconds)
150C
60
D-glucose
0
B
60 xZ
ai100C
5- AI 6-
40 E
E
E
0
a 20 °41cl
-0
7
_
.2c
Q.
0
20
40
60
80
100
120
time (minutes)
FIG. 2. Time course of D-glucose uptake. The reconstituted li(0.7 mg/ml of protein solubilized with Triton X-100 and 15 mg/ml of phospholipids) were prepared as described in Materials and Methods. (A) Rapid uptake of D-glucose. Uptake was started by addition of 0.2 mM D- or L-[14C]glucose to the reaction mixture containing reconstituted liposomes (56 gg of protein and 1.2 mg of phospholipids). At indicated times, it was stopped with a cold stopping solution containing mercuric ions and followed by filtration through a membrane filter. (B) Uptake of D- or L-glucose for longer periods. The same assay conditions as in (A) were used. posomes
A
B
C
FIG. 1. Sodium dodecyl sulfate-gel electrophoresis of ghosts (A), Triton-X-100-solubilized protein fraction (B), and octylglucoside-solubilized protein fraction (C). About 50 ,gg of protein was applied to each gel and stained with Coomassie blue. The tracking dye was bromophenol blue (BPB).
Gel Filtration Method. After addition of 0.3 ml of the stopping solution, the uptake medium was applied to a Sephadex column (G-50 medium, 25 cm X 0.7 cm) and elution was performed at 40 with the stopping solution. It took 15-20 min from the end of uptake until the elution of the
liposome fraction. Radioactivity of each fraction (0.6 ml) counted in Bray's solution (31). Membrane Filter Method. After addition of 5 ml of the stopping solution, the uptake medium was passed through a membrane filter (Selectron 0.2 gm or Millipore 0.22 gm), followed by a 10 ml wash with the cold stopping solution. Radioactivity was counted in Bray's solution. When a proper amount of the reconstituted liposomes (less than 2 mg of phospholipids) was used, the membrane filter method showed more than 80% of D-glucose retention observed with the gel filtration method. Analytical methods Protein (32), phosphorus (33), Triton X-100 (29), and octylglucoside (23) were measured as described. Sodium dodecyl sulfate gel electrophoresis (5% acrylamide, 0.2% sodium dodecyl sulfate) was performed according to the published method (25, 34). Each gel was stained with Coomassie blue G, periodic acid-Schiff reagent (25), or a carbocyanine dye (35). Paper chromatography of sugars was performed using Whatman no. 4 paper and a solvent mixture of n-butanol, ethanol, and water (50:32:18). When the D-glucose uptake in the reconstituted liposomes was assayed with the membrane filter method more than 90% of the radioisotope on the filter co-migrated with D-glucose.
was
RESULTS AND DISCUSSION Properties of the solubilized protein fractions *About half of the protein (47%) was removed from ghosts by the EDTA-NaCl treatment. The washed vesicles were incubated with non-ionic detergents (Triton X-100 or octylglucoside). Both detergents solubilized a considerable amount of protein from the vesicles (70% with Triton X-100 and 56% with octylglucoside). The protein fraction solubilized with Triton X-100 contained several proteins, of which a main protein was band 3 (36), and octylglucoside gave the same result (Fig. 1). Four glycoprotein bands (PAS 1-4) were also observed with periodic acid-Schiff staining or a carbocyanine dye staining (not shown). Phospholipids were also solubilized; 0.39 ,mol of total P per mg of protein with Triton X-100 and 0.56 Amol of total P per mg of protein with octylglucoside. Satisfactory removal of detergents was achieved by the Bio-Beads SM-2 treatment or diafiltration. Ultrafiltration was performed to determine the free detergent concentration in the final protein fractions; the ultrafiltrate contained 0.002% Triton X-100 or 10 WM octylglucoside. These values were well below the critical micelle concentrations of the detergents (23, 29). Characteristics of D-glucose uptake The reconstituted liposomes showed time-dependent, rapid D-glucose uptake, with a half time of 6 sec (Fig. 2A). To estimate the nonspecific transport and/or binding, L-glucose, which is not a substrate for the monosaccharide transport system in human erythrocytes (1), was used. The uptake of L-glucose showed a slower time course. The initial rate of uptake was 36 times faster for D-glucose than for L-glucose.
898
Proc. Nat. Acad. Sci. USA 73 (1976)
Biochemistry: Kasahara and Hinkle
Table 1. Substrate specificity of the reconstituted sugar. uptake system
Uptake (percent of total radioisotope/15 sec) Fraction of*
rapid Addition
D-Glucose
L-Glucose
D-glucose uptake
D-Glucose 2-Deoxy-D-glucose 3-O-Methyl-D-glucose D-Mannose D-Galactose D-Ribose L-Glucose
0.054 0.022 0.027 0.025 0.025 0.027 0.048 0.048
0.012 0.009 0.009 0.009 0.010 0.009 0.010 0.011
0.042 0.013 0.018 0.016 0.015 0.018 0.038 0.037
Percent inhibition 69 57 62 64 57 10 12
Each sugar (20 mM) with 0.2 mM D- or L-[14C]glucose was added to the reaction mixture containing reconstituted vesicles (86,gg of protein solubilized with Triton X-100 and 1.2 mg of phospholipids) to start the uptake. Uptake was measured with the membrane filter method. * Fraction of rapid D-glucose uptake was calculated by subtracting L-glucose uptake from D-glucose uptake.
After the rapid phase, uptake of D-glucose showed a slow phase which continued for about 2 hr (Fig. 2B). Uptake of L-glucose showed only the slow phase with a half time of about 30 min and appeared to approach the same final extent as D-glucose uptake. At zero time, a small apparent uptake of both isomers was observed. Since liposomes without protein showed a slow uptake of both isomers and similar zero time uptake (not shown), the rapid uptake of D-glucose shown in Fig. 2A is the component dependent on the protein fraction. The slow uptake of both isomers in reconstituted Iiposomes may indicate penetration via simple diffusion and the zero time uptake may result from nonspecific retention of isotopes. The extent of the rapid D-glucose uptake (30 see) corresponded to 48% of the total extent (2 hr) in the experiment shown in Fig. 2, but this value showed considerable Table 2. Effect of inhibitors on the reconstituted system
Uptake, pmol/mg of protein Substrate
Addition
per 15 sec
Exp. 1* 113
D-Glucose
L-Glucose
HgCl2, 20,.iM HgCl2, 600 pM HgCl2, 600 PM
D-Glucose
Exp. 2 Ethanol, 1%
Cytochalasin Bt, 1 ,uMt Cytochalasin B, 5 JM L-Glucose
Ethanol, 1%
variation from 18% to 60%, in other experiments. No signifidifference of uptake activity was found between the Triton-X-100- and octylglucoside-solubilized protein fractions.
cant
The substrate specificity of the D-glucose uptake system studied by inhibition with nonradioactive sugars (Table 1). D-Glucose uptake was inhibited by several sugars (D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, D-mannose, D-galactose) which are known to have affinity for the monosaccharide transport system in erythrocytes (1, 37). L-Glucose and D-ribose did not inhibit well, as expected from the results on the erythrocytes. D-Glucose uptake was inhibited by two reagents. Table 2 shows that mercuric ions and cytochalasin B inhibited the uptake at the concentration comparable to that for erythrocytes (38-40). Stilbestrol, which inhibits monosaccharide transport in erythrocytes and ghosts (1, 41), did not inhibit the reconstituted system even at 50 ,uM. This might indicate that some factors are missing in the present reconstituted
was
system.
The possibility that the results reflect binding instead of ways. The uptake was de-
transport was examined in several
Table 3. Effect of lipid and protein
34 34 25 49
123 82 51 38 52 37
Cytochalasin B, 1 JM Cytochalasin B, 5 ,M The Triton-X-100-solubilized protein fraction was used for the reconstitution. Phospholipid concentration after sonication was 15. mg/ml (Exp. 1) or 20 mg/ml (Exp. 2) and that of protein was 0.75 mg/ml (Exp. 1) or 0.80 mg/ml (Exp. 2). Uptake was measured with the gel filtration method (Exp. 1) or the membrane filter method (Exp. 2). * Uptake was measured at 100. t Cytochalasin B was dissolved in ethanol and mixed in the assay mixture. Maximum ethanol concentration in the assay was 1%.
on
D-glucose uptake
Uptake (percent of total radioisotope)
Reconstituted liposomes* Protein alonet Liposomes alone t
D-Glucose
L-Glucose
0.110 0.004 0.020
0.037 0.026
The uptake was measured with the gel filtration method for 1 min. * The reconstituted liposomes (15 mg/ml of phospholipids and 0.75 mg/ml of protein solubilized with Triton X-100) were prepared as described in Materials and Methods. A portion of 140 gl (0.1 mg of protein and 2.1 mg of phospholipids) was used for each uptake assay. t "Protein alone" was prepared by adjusting the KCI concentration of the protein fraction to 150 mM and 0.1 mg of protein was used for each assay. "Liposomes alone" was prepared in the same way as "Reconstituted liposomes" except that Tris-KCI mixture was added in place of protein and 2.1 mg of phospholipids was used for each assay.
Biochemistry:
Kasahara and Hinkle
004|-\
D-glucose
003 o
L-glucose
0
Proc. Nat. Acad. Sci. USA 73 (1976)
We wish to thank Dr. L. A. Heppel, who was associated with this research, for many discussions and continual encouragement. We are also grateful to Dr. E. Racker for providing octylglucoside and stimulating discussions and criticisms. This study was supported by National Institutes of Health Grant no. CA-14454. 1. LeFevre, P. G. (1961) Pharmacol. Rev. 13, 39-70. 2. Stein, W. D. (1967) in The Movement of Molecules Across Cell Membranes (Academic Press, New York), pp. 126-176 and pp. 266-308.
0.02
C1
0.01 _ o
0
5
10
15
sonication time (minutes)
FIG. 3. Dependence of the reconstitution on sonication time. Liposomes were mixed with the Triton-X-100-solubilized protein fraction (15 mg/ml of phospholipids and 0.59 mg/ml of protein) and sonicated for the indicated time at 350. A portion of 120 ,u was used for a 15 sec uptake assay by the membrane filter method.
pendent
399
lipid
on both and was essential for
the protein fraction (Table 3). the reconstitution (Fig. 3), but sonication times longer than 3 min were inhibitory. The liposomes showed loss of D-['4C]glucose when chased with nonradioactive D-glucose (Fig. 4). The final extent of D['4C]glucose uptake in the liposomes returned almost to the original level after this transient loss. Triton X-100 (0.5%) or Tween 80 (0.5%) inhibited the D-glucose uptake (30 sec) by 89% and 81%, respectively (not shown). At a higher concentration of D-glucose (20 mM), the initial rate of uptake was 4.5 nmol/mg of protein per 10 sec and the extent of D-glucose taken up reached 14 nmol/mg of protein at 5 min (not shown). This extent is 3.5 times higher than the upper limit of binding capacity in the protein fraction if we assume that one protein molecule binds maximally one sugar molecule, and that 40% of the protein fraction, which corresponds to the maximum fraction of a single protein (band 3), is the glucose binding protein and the molecular weight of the protein is 100,000. These results are consistent with the idea that the uptake of D-glucose is transport of the sugar into the reconstituted liposomes via a specific transport system.. The isolation of a transport system and reconstitution into a well-defined membrane are indispensable for the study of transport at the molecular level. The present work is a first step toward such studies of the monosaccharide transport system of human erythrocytes.
Sonication
FIG. 4. Transient loss of accumulated radioactive D-glucose induced by addition of nonradioactive D-glucose. At 30 sec, nonradioactive 10 mM D- or L-glucose was added to the assay mixture (closed symbols). The assay mixture contained reconstituted liposomes (56 /g of protein solubilized with Triton X-100 and 1.2 mg of phospholipids). Uptake was measured by the membrane filter method.
3. Kotyk, A. (1975) in Cell Membrane Transport. Principles and Techniques, eds. Kotyk, A. & Janfacek, K. (Plenum Press, New York), pp. 391-395. 4. Lieb, W. R. & Stein, W. D. (1972) Biochim. Biophys. Acta 265, 187-207. 5. LeFevre, P. G. (1973) J. Membr. Biol. 11, 1-19. 6. Lin, S. & Spudich, J. A. (1974) Biochem. Biophys. Res. Commun. 61, 1471-1476. 7. Taverna, R. D. & Langdon, R. G. (1973) Biochem. Biophys. Res. Commun. 54,593-599. 8. Jung, C. Y. & Carlson, L. M. (1975) J. Biol. Chem. 250, 3217-3220. 9. Steck, T. L. (1974) J. Cell Biol. 62, 1-19. 10. Wood, R. E., Wirth, F. P., Jr. & Morgan, H. E. (1968) Biochim. Biophys. Acta 163, 171-178. 11. Jung, C. Y., Chaney, J. E. & LeFevre, P. G. (1968) Arch. Biochem. Biophys. 126,664-676. 12. LeFevre, P. G., Jung, C. Y. & Chaney, J. E. (1968) Arch. Biochem. Biophys. 126,677-691. 13. Jung, C. Y. (1971) J. Membr. Biol. 5,200-214. 14. Lidgard, G. P. & Jones, M. N. (1975) J. Membr. Biol. 21, 1-10.
15. LeFevre, P. G. & Masiak, S. J. (1970) J. Membr. Biol. 3,387399. 16. Kagawa, Y. & Racker, E. (1971) J. Biol. Chem. 246, 54775487. 17. Hinkle, P. C., Kim, J. J. & Racker, E. (1972) J. Biol. Chem. 247, 1338-1339. 18. Racker, E. (1972) J. Biol. Chem. 247, 8198-8200. 19. Racker, E. & Stoeckenius, W. (1974) J. Biol. Chem. 249, 662-663. 20. Goldin, S. M. & Tong, S. W. (1974) J. Biol. Chem. 249, 5907-5915. 21. Sb-rtzer, H. G. & Racker, E. (1974) J. Biol. Chem. 249, 1320-1321. 22. Rothstein, A., Cabantchik, Z. I., Balshin, M. & Juliano, R. (1975) Biochem. Biophys. Res. Commun. 64, 144-150. 23. Baron, C. & Thompson, T. E. (1975) Biochim. Biophys. Acta 382,276-285. 24. Dodge, J. T., Mitchell, C. & Hanahan, D. J. (1963) Arch. Biochem. Biophys. 100, 119-130. 25. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971) Biochemistry 10, 2606-2617. 26. Privett, 0. S., Lundberg, W. O., Khan, N. A., Tolberg, W. E. & Wheeler, D. H. (1953) J. Am. Oil Chem. Soc. 30,61-66. 27. Witheiler, A. P. (1971) Doctoral Dissertation, Cornell University. 28. Wagner, C. D., Clever, H. L. & Peters, E. D. (as described in) Johnson, R. M. & Siddiqi, I. W. (1970) The Determination of Organic Peroxides (Pergamon Press, Oxford), p. 46. 29. Holloway, P. W. (1973) Anal. Biochem. 53,304-308. 30. Racker, E. (1973) Biochem. Biophys. Res. Commun. 55, 224-230. 31. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 32. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 33. Ames, B. N. & Dubin, D. T. (1960) J. Biol. Chem. 235, 769775. 34. Steck, T. L. & Yu, J. (1973) J. Supramol. Struct. 1, 220-232. 35. Green, M. R. & Pastewka, J. V. (1975) Anal. Biochem. 65, 66-72.
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36. Yu, J., Fischman, D. A. & Steck, T. L. (1973) J. Supramol. Struct. 1,233-248. 37. Barnett, J. E. G., Holman, G. D. & Munday, K. A. (1973) Biochem. J. 131,211-221. 38. LeFevre, P. G. (1948) J. Gen. Physiol. 31, 505-527.
Proc. Nat. Acad. Sci. USA 73 (1976) 39. Bloch, R. (1973) Biochemistry 12,4799-4801. 40. Taverna, R. D. & Langdon, R. G. (1973) Biochim. Biophys. Acta 323,207-219. 41. Jung, C. Y., Carlson, L. M. & Whaley, D. A. (1971) Biochim. Biophys. Acta 241, 613-627.
