Eur. J. Biochem. 80,425-432 (1977)
Characterization of the Cellular Binding of Exogenous Gangliosides Richard CALLIES, Gunter SCHWARZMANN, Klaus RADSAK, Rudolf SIEGERT, and Herbert WIEGANDT Hygiene Institut and Physiologisch-Chemisches Institut I , Philipps-Universitat Marburg (Received March 22, 1977)
Experiments were performed to characterize the accumulation of different exogenous sialoglycolipids by cells. A biphasic kinetics of accumulation appeared to be characteristic for intact primary and permanent monolayer cells and chicken erythrocytes whereas that of chicken erythrocyte ghosts was monophasic. Saturation levels could be reached with Galbl+3 GalNAcpl+4 Gal[3+2aNeuAc],!I1 +4 GlcPl - 1’Cer and Galfil-+3 GalNAcfil -4 Ga1[3c2aNeuAc8+2aNeuAc]fil+4 Glc@ 1’Cer with concentrations of 100 and 200 nmol per ml culture medium, respectively. Under these conditions the amount of 1.3- 1.4 nmol of sialoglycolipids per 100 pg of cellular protein were found to be cell-bound after approximately two hours. In case of the ganglioside NeuAccr2+3GalfiI -4 Glcfll - 1’Cer no saturation could be reached. The intermediate plateau of accumulation was abolished by the agents colchicine and sodium fluoride which on the other hand had no influence on the level of saturation. In contrast the presence of cytochalasin B, 2-deoxy-~-glucoseplus sodium azide, and glutardialdehyde as well as low temperature decreased the amount of incorporated gangliosides drastically. The effect of cytochalasin B was limited to monolayer cultures. Mild trypsinisation liberated some 70 - 85 % of cell-associated gangliosides even after druginhibited accumulation of gangliosides. Also some 87 - 95 % of the cell-bound ganglioside NeuAca2+3 Galpl -4 Glcpl - 1‘Cer remained accessible to the action of exogenous sialidase, thus excluding gross internalisation Over the last several years reports have been accumulating that glycosphingolipids as constituents of the outer surface of mammalian cell membranes significantly contribute by means of their carbohydrate structures to a variety of cellular events. Since glycolipids are taken up by cells from the culture medium, attempts were reported to supplement surface membrane glycolipids by incorporation of exogenous compounds [I]. Glycolipid uptake was accompanied by a reduction in growth rate and saturation density [2- 61. Abbreviations. Abbreviations were used according to principles outlined earlier Wiegandt, H. (1973) Hoppe-Seyler’s Z . Physiol. Chetn. 354, 3049- 1056. The first name given is that used in the text (for simplification the term NeuAc has been deleted in the text), the third name is designated as recommended by IUPAC-IUB Lipid Nomenclature, 1976, the final designation is according to Svennerholm (1963) J . Neurochem. 10, 613-623. GL,,lNeuAc = (I13NeuAc-Lac-Cer) = NeuAcct2-3 Gdlpl -t4 Glc,!?l- 1’Cer = GM3; GGtetlNeuAc= (I13NeuAc-CgOse4-Cer) = Galp1+3 GalNAcfil-4 Gdl[3t2aNeuAc]fi1+4 GlcPl - 1’Cer = GMl; Gct,,2bNeuAc = I13(NeuAc)z-GgOse4-Cer = Galpl-3 GalNAcPl-4 Gal[3+2aNeuAc8t2aNeuAc]pl+4 Glcpl- 1‘Cer = GDlh. Enzymes. Trypsin (EC 3.4.21.4); galactose oxidase or D-galactose: oxygen 6-oxidoreductase (EC 1.1.3.9).
A functional incorporation of ganglioside into cells to serve as receptor molecules for cholera toxin was reported from a number of laboratories [7- 101. The interaction with the toxin thereby causes the incorporated lipid to move in the plane of the membranes causing an apparent ganglioside capping phenomenon [I 1- 131. The underlying mechanisms, however, by which these effects are produced are not understood. Therefore an attempt was made to characterize the mode of accumulation of exogenous glycosphingolipids by the cell.
MATERIALS AND METHODS Muter iuls Sodium azide, sodium fluoride, sodium borohydride, dimethylsulphoxide (all analytical grade) and silica gel G coated thin-layer plates were purchased from Merck AG (Darmstadt, F.R.G.). Colchicine, glutardialdehyde and penicillin were from Serva
Ganglioside Incorporation
426
(Heidelberg, F.R.G.). Szintigel and calf serum were obtained from Roth (Karlsruhe, F.R.G.). Cytochalasin B was purchased from Aldrich (Milwaukee, Wis., U.S.A.). Trypsin was obtained from Difco (Detroit, Mich., U.S.A.). Vibrio cholera neuraminidase was a generous gift from Behring Werke AG (Marburg, F.R.G.). Galactose oxidase was from Sigma Chemical Co. (St Louis, Mo., U.S.A.) and potassium b ~ r o [ ~ H ] hydride from Amersham-Buchler (Braunschweig, F. R.G.) . Cells Most of the experiments were performed with a permanent mouse cell line (Cl-1-D [14]). As primary cultures chicken embryo fibroblasts were used [15]. Cells were grown on 8 x 28-mm cover slips in Leighton tubes with 2 ml of Eagle's medium supplemented with 5 % calf serum and 200 I.U. penicillin/ml. At confluency one cover slip corresponded to approximately 2 x lo5 cells or 180-200 pg of cellular protein. Chicken erythrocytes were prepared from fresh citrated blood by centrifugation at 2000 x g for 10 min and three subsequent washes in phosphate-buffered saline (NaC1/Pi). Preparation of Erythrocyte Ghosts
Erythrocyte ghosts were isolated from fresh chicken erythrocytes by the method of Dodge [16] with the modification as described by Rosenberg [17]. Preparation and Labeling of Gangliosides
Gangliosides GGtetland Gctet2bwere from normal human brain [18]. GGtetlwas especially purified using the procedure described by Hakomori [19]. GLacl was isolated from normal human spleen by extraction into chloroform/methanol. The chloroform/methanol solution was evaporated to dryness in vacuo and the residue taken up in toluene. The glycolipids were then precipitated by stirring the toluene dispersion into a 30-fold volume of dry acetone. After further purification as described [19] the GLa,l was separated from the neutral glycosphingolipids using the procedure of Yu and Ledeen [20]. Gctet2bwas made radioactive by oxidation of the terminal galactose with galactose oxidase and reduction with tritiated sodium borohydride [21]. The resultant product had a specific activity as described [l]. G L and ~ GGtetl ~ ~ were labeled with tritium using tritiated potassium borohydride (unpublished results). The resultant radioactive gangliosides GGtetl and G L had ~ specific ~ ~ activities which exceeded lo6 dis. x min-' x nmol-'. Gangliosides were evaporated from chloroform/methanol in sterile flasks under a stream of nitrogen and
directly dissolved under sterile conditions in culture medium of the desired concentration under slight warming and shaking. Incubation of Cells with Gangliosides
Prior to incubation of the cover slip cultures in the Leighton tubes it was essential to remove residues of serum of the growth medium by extensive washings with Eagle's medium without calf serum, as serum had been observed in preliminary experiments to interfere with ganglioside accumulation by the cells (unpublished results). Cover slips were then exposed at 37 "C to Eagle's medium less calf serum supplemented with various concentrations of radioactive ganglioside for the intervals indicated in Results. Following incubation the cover slips were removed from the Leighton tubes and washed be repeated dipping in NaCl/Pi. The washing procedure had been established in such a way that no further decline in cell-associated radioactivity could be observed by additional washings. Washed chicken erythrocytes and ghosts, prepared from an equivalent number of cells, were resuspended at a concentration of 4 x lo7 cells/ml Eagle's medium containing radioactive ganglioside and incubated with stirring in a 37 "C water bath. Following incubation erythrocytes as well as ghosts were washed by repeated sedimentation and resuspension in NaC1/Pi. Trypsinisation of Cells
Mild trypsinisation before or after ganglioside accumulation was carried out with 0.25 % trypsin in NaC1/Pi, pH 7.8 for 20 min at 37 "C. The detached cells were subsequently washed extensively by sedimentation and resuspension in NaCl/Pi at 4 "C. For comparison identical monolayer cultures were detached by EDTA (1 mM in NaCl/Pi) and used in corresponding experiments. Cover slip cultures as well as cells detached by trypsin or EDTA were found viable to more than 95 % as determined with the dye exclusion method using trypan blue. Reduction with Sodium Borohydride
Reduction of the reaction products of cells preincubated with glutardialdehyde and extensively washed was carried out using sodium borohydride as described by Baily [22]. Liquid Scintillation Counting
For the determination of cell-associated radioactivity as well as liberated radioactivity in aqueous solution, cells and aliquots of the supernatants were directly mixed with a detergent containing toluene-
427
R. Callies, G. Schwarzmann, K. Radsak, R. Siegert, and H. Wiegandt
Fig. 1. Kinetics qf binding of'gangliosides hy Cl-I-D cells at 37 C. (m......) GGtetl,127 nmol/ml incubation medium; (-.-). GL,,~, 169 nmol/ml incubation medium; (A----A) GL,,~,42.5 nmol/ml incubation medium. Cover slip cultures were incubated with Eagle's medium containing 3H-labeled ganglioside and cell-associated radioactivity determined after the indicated intervals
based scintillation fluid (Szintigel, Roth, F.R.G.) and counted in a Beckman liquid scintillation counter (model LS 230). Additional experiments revealed that the trypsin released radioactivity could not be sedimented by centrifugation at 100000 x g for one hour. Amounts of gangliosides incorporated or liberated were calculated from the measured and specific radioactivities of the ganglioside solutions used for incubation experiments. Each value presented in Results refers to the mean of determinations derived from at least five identically treated samples. Protein Determination
Protein was determined by the method of Lowry et al. [23] with bovine serum albumin as a standard.
described by van den Eijnden [24] the thin-layer plates were cut into strips. After visualisation of the bands using the Ehrlich spray [25] and the orcinol spray [26] the appropriate bands of parallel strips were cut out and counted directly in a toluene-based scintillation fluid using an LKB-Wallac liquid scintillation counter.
RESULTS For cell incorporation studies the gangliosides GLacl, GGtetl and Gctet2bwere labeled by tritiation of the carbon-carbon double bond with sodium borotritiide. By use of these gangliosides, whose specific activity by far exceeded those which had been used previously [l], the kinetics of uptake by different cell systems was reexamined.
Treatment of Cells with Neuraminidase
Cover slip cultures following incubation with GL,,l were exposed to 50 units of Vibrio cholera neuraminidase in 1 ml Eagle's medium for 1 h at 37 "C. Subsequently the cover slips were extensively washed as described above. Determination of the Conversion of G L , , ~by Neuraminidase
For the determination of the degree of conversion of cell associated GLa,l to lactosylceramide by the action of neuraminidase the labeled ganglioside was recovered by extracting the cells with chloroform/ methanol. Aliquots as such and mixed with unlabeled GL,,1 and lactosylceramide derived from GL~,Iby mild acid hydrolysis in 0.1 M HCI at 60 "C for one hour were spotted on silica gel G thin-layer plates. After development using the solvent mixtures as
Accumulation of Gangliosides by Cells
All three gangliosides are being accumulated with time, whereby a plateau of incorporation is reached after some 5 - 15 min. It persisted for approximately 10 min when the uptake again increased to reach a final stage after approximately 2 h. Thereafter no significant further uptake of glycolipids was noticed (Fig. 1). In contrast to intact erythrocytes their corresponding cell ghosts did not show the intermediate plateau of saturation during uptake of gangliosides (Fig. 2). With the gangliosides GGtetland Gctet2b,a saturation of binding of about 1.4 nmol ganglioside per 100 yg total cellular protein was reached (Fig. 3). Thereby these two gangliosides could compete with each other for incorporation as had been shown earlier [I]. Despite the fact, that ganglioside GL,,~at various concentrations paralleled the other gangliosides in
Ganglioside Incorporation
428 17
16 15 14 (II
c Y)
0
L m
K I
-.'.'
-
m c m
cl 1
0
10
20
30
60
40
90
12c
Incubation time (min)
Fig. 2. Kinetics of binding of gangliuside Gctetlby erythrocytes and erythrocyte ghosts at 37 "C. Erythrocytes and ghosts were incubated with stirring in Eagle's medium containing 3H-labeledgangliosides (127 nmoliml), and bound radioactivity determined after the indicated intervals. (@-.-.-@) Erythrocytes, (=---.)erythrocyte ghosts. Results were expressed as nmol/mg by taking 4 x lo7 erythrocytes as corresponding to 1 mg cellular protein, and erythrocyte ghost preparation corresponding to 4 x lo7 erythrocytes 3 -
G a n g l i o s i d e (nrnol/rnl)
Fig.3. Binding of G ~ t ~( tl Gc,,,Zb I (A-A)= und C;L,,I (@d) ) by Cl-I-D, cells as afunction qf.ganglioside concentration in the incubation medium. Cover slip cultures were incubated with Eagle's medium containing different concentrations of 3H-labeled ganglioside at 37 "C for 2 h at which time cell-associated radioactivity was determined 3.0 I-
2.5 -
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c a ,
2
5 2.0
%2 gz 1.5 -
.-
-
I
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........................
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Characterization of the Cellular Binding of Exogenous Gangliosides Richard CALLIES, Gunter SCHWARZMANN, Klaus RADSAK, Rudolf SIEGERT, and Herbert WIEGANDT Hygiene Institut and Physiologisch-Chemisches Institut I , Philipps-Universitat Marburg (Received March 22, 1977)
Experiments were performed to characterize the accumulation of different exogenous sialoglycolipids by cells. A biphasic kinetics of accumulation appeared to be characteristic for intact primary and permanent monolayer cells and chicken erythrocytes whereas that of chicken erythrocyte ghosts was monophasic. Saturation levels could be reached with Galbl+3 GalNAcpl+4 Gal[3+2aNeuAc],!I1 +4 GlcPl - 1’Cer and Galfil-+3 GalNAcfil -4 Ga1[3c2aNeuAc8+2aNeuAc]fil+4 Glc@ 1’Cer with concentrations of 100 and 200 nmol per ml culture medium, respectively. Under these conditions the amount of 1.3- 1.4 nmol of sialoglycolipids per 100 pg of cellular protein were found to be cell-bound after approximately two hours. In case of the ganglioside NeuAccr2+3GalfiI -4 Glcfll - 1’Cer no saturation could be reached. The intermediate plateau of accumulation was abolished by the agents colchicine and sodium fluoride which on the other hand had no influence on the level of saturation. In contrast the presence of cytochalasin B, 2-deoxy-~-glucoseplus sodium azide, and glutardialdehyde as well as low temperature decreased the amount of incorporated gangliosides drastically. The effect of cytochalasin B was limited to monolayer cultures. Mild trypsinisation liberated some 70 - 85 % of cell-associated gangliosides even after druginhibited accumulation of gangliosides. Also some 87 - 95 % of the cell-bound ganglioside NeuAca2+3 Galpl -4 Glcpl - 1‘Cer remained accessible to the action of exogenous sialidase, thus excluding gross internalisation Over the last several years reports have been accumulating that glycosphingolipids as constituents of the outer surface of mammalian cell membranes significantly contribute by means of their carbohydrate structures to a variety of cellular events. Since glycolipids are taken up by cells from the culture medium, attempts were reported to supplement surface membrane glycolipids by incorporation of exogenous compounds [I]. Glycolipid uptake was accompanied by a reduction in growth rate and saturation density [2- 61. Abbreviations. Abbreviations were used according to principles outlined earlier Wiegandt, H. (1973) Hoppe-Seyler’s Z . Physiol. Chetn. 354, 3049- 1056. The first name given is that used in the text (for simplification the term NeuAc has been deleted in the text), the third name is designated as recommended by IUPAC-IUB Lipid Nomenclature, 1976, the final designation is according to Svennerholm (1963) J . Neurochem. 10, 613-623. GL,,lNeuAc = (I13NeuAc-Lac-Cer) = NeuAcct2-3 Gdlpl -t4 Glc,!?l- 1’Cer = GM3; GGtetlNeuAc= (I13NeuAc-CgOse4-Cer) = Galp1+3 GalNAcfil-4 Gdl[3t2aNeuAc]fi1+4 GlcPl - 1’Cer = GMl; Gct,,2bNeuAc = I13(NeuAc)z-GgOse4-Cer = Galpl-3 GalNAcPl-4 Gal[3+2aNeuAc8t2aNeuAc]pl+4 Glcpl- 1‘Cer = GDlh. Enzymes. Trypsin (EC 3.4.21.4); galactose oxidase or D-galactose: oxygen 6-oxidoreductase (EC 1.1.3.9).
A functional incorporation of ganglioside into cells to serve as receptor molecules for cholera toxin was reported from a number of laboratories [7- 101. The interaction with the toxin thereby causes the incorporated lipid to move in the plane of the membranes causing an apparent ganglioside capping phenomenon [I 1- 131. The underlying mechanisms, however, by which these effects are produced are not understood. Therefore an attempt was made to characterize the mode of accumulation of exogenous glycosphingolipids by the cell.
MATERIALS AND METHODS Muter iuls Sodium azide, sodium fluoride, sodium borohydride, dimethylsulphoxide (all analytical grade) and silica gel G coated thin-layer plates were purchased from Merck AG (Darmstadt, F.R.G.). Colchicine, glutardialdehyde and penicillin were from Serva
Ganglioside Incorporation
426
(Heidelberg, F.R.G.). Szintigel and calf serum were obtained from Roth (Karlsruhe, F.R.G.). Cytochalasin B was purchased from Aldrich (Milwaukee, Wis., U.S.A.). Trypsin was obtained from Difco (Detroit, Mich., U.S.A.). Vibrio cholera neuraminidase was a generous gift from Behring Werke AG (Marburg, F.R.G.). Galactose oxidase was from Sigma Chemical Co. (St Louis, Mo., U.S.A.) and potassium b ~ r o [ ~ H ] hydride from Amersham-Buchler (Braunschweig, F. R.G.) . Cells Most of the experiments were performed with a permanent mouse cell line (Cl-1-D [14]). As primary cultures chicken embryo fibroblasts were used [15]. Cells were grown on 8 x 28-mm cover slips in Leighton tubes with 2 ml of Eagle's medium supplemented with 5 % calf serum and 200 I.U. penicillin/ml. At confluency one cover slip corresponded to approximately 2 x lo5 cells or 180-200 pg of cellular protein. Chicken erythrocytes were prepared from fresh citrated blood by centrifugation at 2000 x g for 10 min and three subsequent washes in phosphate-buffered saline (NaC1/Pi). Preparation of Erythrocyte Ghosts
Erythrocyte ghosts were isolated from fresh chicken erythrocytes by the method of Dodge [16] with the modification as described by Rosenberg [17]. Preparation and Labeling of Gangliosides
Gangliosides GGtetland Gctet2bwere from normal human brain [18]. GGtetlwas especially purified using the procedure described by Hakomori [19]. GLacl was isolated from normal human spleen by extraction into chloroform/methanol. The chloroform/methanol solution was evaporated to dryness in vacuo and the residue taken up in toluene. The glycolipids were then precipitated by stirring the toluene dispersion into a 30-fold volume of dry acetone. After further purification as described [19] the GLa,l was separated from the neutral glycosphingolipids using the procedure of Yu and Ledeen [20]. Gctet2bwas made radioactive by oxidation of the terminal galactose with galactose oxidase and reduction with tritiated sodium borohydride [21]. The resultant product had a specific activity as described [l]. G L and ~ GGtetl ~ ~ were labeled with tritium using tritiated potassium borohydride (unpublished results). The resultant radioactive gangliosides GGtetl and G L had ~ specific ~ ~ activities which exceeded lo6 dis. x min-' x nmol-'. Gangliosides were evaporated from chloroform/methanol in sterile flasks under a stream of nitrogen and
directly dissolved under sterile conditions in culture medium of the desired concentration under slight warming and shaking. Incubation of Cells with Gangliosides
Prior to incubation of the cover slip cultures in the Leighton tubes it was essential to remove residues of serum of the growth medium by extensive washings with Eagle's medium without calf serum, as serum had been observed in preliminary experiments to interfere with ganglioside accumulation by the cells (unpublished results). Cover slips were then exposed at 37 "C to Eagle's medium less calf serum supplemented with various concentrations of radioactive ganglioside for the intervals indicated in Results. Following incubation the cover slips were removed from the Leighton tubes and washed be repeated dipping in NaCl/Pi. The washing procedure had been established in such a way that no further decline in cell-associated radioactivity could be observed by additional washings. Washed chicken erythrocytes and ghosts, prepared from an equivalent number of cells, were resuspended at a concentration of 4 x lo7 cells/ml Eagle's medium containing radioactive ganglioside and incubated with stirring in a 37 "C water bath. Following incubation erythrocytes as well as ghosts were washed by repeated sedimentation and resuspension in NaC1/Pi. Trypsinisation of Cells
Mild trypsinisation before or after ganglioside accumulation was carried out with 0.25 % trypsin in NaC1/Pi, pH 7.8 for 20 min at 37 "C. The detached cells were subsequently washed extensively by sedimentation and resuspension in NaCl/Pi at 4 "C. For comparison identical monolayer cultures were detached by EDTA (1 mM in NaCl/Pi) and used in corresponding experiments. Cover slip cultures as well as cells detached by trypsin or EDTA were found viable to more than 95 % as determined with the dye exclusion method using trypan blue. Reduction with Sodium Borohydride
Reduction of the reaction products of cells preincubated with glutardialdehyde and extensively washed was carried out using sodium borohydride as described by Baily [22]. Liquid Scintillation Counting
For the determination of cell-associated radioactivity as well as liberated radioactivity in aqueous solution, cells and aliquots of the supernatants were directly mixed with a detergent containing toluene-
427
R. Callies, G. Schwarzmann, K. Radsak, R. Siegert, and H. Wiegandt
Fig. 1. Kinetics qf binding of'gangliosides hy Cl-I-D cells at 37 C. (m......) GGtetl,127 nmol/ml incubation medium; (-.-). GL,,~, 169 nmol/ml incubation medium; (A----A) GL,,~,42.5 nmol/ml incubation medium. Cover slip cultures were incubated with Eagle's medium containing 3H-labeled ganglioside and cell-associated radioactivity determined after the indicated intervals
based scintillation fluid (Szintigel, Roth, F.R.G.) and counted in a Beckman liquid scintillation counter (model LS 230). Additional experiments revealed that the trypsin released radioactivity could not be sedimented by centrifugation at 100000 x g for one hour. Amounts of gangliosides incorporated or liberated were calculated from the measured and specific radioactivities of the ganglioside solutions used for incubation experiments. Each value presented in Results refers to the mean of determinations derived from at least five identically treated samples. Protein Determination
Protein was determined by the method of Lowry et al. [23] with bovine serum albumin as a standard.
described by van den Eijnden [24] the thin-layer plates were cut into strips. After visualisation of the bands using the Ehrlich spray [25] and the orcinol spray [26] the appropriate bands of parallel strips were cut out and counted directly in a toluene-based scintillation fluid using an LKB-Wallac liquid scintillation counter.
RESULTS For cell incorporation studies the gangliosides GLacl, GGtetl and Gctet2bwere labeled by tritiation of the carbon-carbon double bond with sodium borotritiide. By use of these gangliosides, whose specific activity by far exceeded those which had been used previously [l], the kinetics of uptake by different cell systems was reexamined.
Treatment of Cells with Neuraminidase
Cover slip cultures following incubation with GL,,l were exposed to 50 units of Vibrio cholera neuraminidase in 1 ml Eagle's medium for 1 h at 37 "C. Subsequently the cover slips were extensively washed as described above. Determination of the Conversion of G L , , ~by Neuraminidase
For the determination of the degree of conversion of cell associated GLa,l to lactosylceramide by the action of neuraminidase the labeled ganglioside was recovered by extracting the cells with chloroform/ methanol. Aliquots as such and mixed with unlabeled GL,,1 and lactosylceramide derived from GL~,Iby mild acid hydrolysis in 0.1 M HCI at 60 "C for one hour were spotted on silica gel G thin-layer plates. After development using the solvent mixtures as
Accumulation of Gangliosides by Cells
All three gangliosides are being accumulated with time, whereby a plateau of incorporation is reached after some 5 - 15 min. It persisted for approximately 10 min when the uptake again increased to reach a final stage after approximately 2 h. Thereafter no significant further uptake of glycolipids was noticed (Fig. 1). In contrast to intact erythrocytes their corresponding cell ghosts did not show the intermediate plateau of saturation during uptake of gangliosides (Fig. 2). With the gangliosides GGtetland Gctet2b,a saturation of binding of about 1.4 nmol ganglioside per 100 yg total cellular protein was reached (Fig. 3). Thereby these two gangliosides could compete with each other for incorporation as had been shown earlier [I]. Despite the fact, that ganglioside GL,,~at various concentrations paralleled the other gangliosides in
Ganglioside Incorporation
428 17
16 15 14 (II
c Y)
0
L m
K I
-.'.'
-
m c m
cl 1
0
10
20
30
60
40
90
12c
Incubation time (min)
Fig. 2. Kinetics of binding of gangliuside Gctetlby erythrocytes and erythrocyte ghosts at 37 "C. Erythrocytes and ghosts were incubated with stirring in Eagle's medium containing 3H-labeledgangliosides (127 nmoliml), and bound radioactivity determined after the indicated intervals. (@-.-.-@) Erythrocytes, (=---.)erythrocyte ghosts. Results were expressed as nmol/mg by taking 4 x lo7 erythrocytes as corresponding to 1 mg cellular protein, and erythrocyte ghost preparation corresponding to 4 x lo7 erythrocytes 3 -
G a n g l i o s i d e (nrnol/rnl)
Fig.3. Binding of G ~ t ~( tl Gc,,,Zb I (A-A)= und C;L,,I (@d) ) by Cl-I-D, cells as afunction qf.ganglioside concentration in the incubation medium. Cover slip cultures were incubated with Eagle's medium containing different concentrations of 3H-labeled ganglioside at 37 "C for 2 h at which time cell-associated radioactivity was determined 3.0 I-
2.5 -
I
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c a ,
2
5 2.0
%2 gz 1.5 -
.-
-
I
~
F Z
,/'
-:1.0.i
...
........................
-----*
/
//-'
,,,'
,A
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0
. . . ,.,,
