567
Biochimica et Biophysica Acta, 497 (1977) 567--577 © Elsevier/North-Holland Biomedical Press
BBA 28224 RAPIDLY M E T A B O L I Z E D G L Y C O P R O T E I N S IN A N E U R O B L A S T O M A CELL LINE
JAMES E. HUDSON and TERRY C. JOHNSON Department o f Microbiology-Immunology, The Medical and Dental Schools, Northwestern University, Chicago, Ill. 60611 (U.S.A.)
(Received November 24th, 1976) Summary The metabolism of neuroblastoma cell glycoproteins was examined using L-[3H]fucose. Incubation of m o n o l a y e r cultures with [3H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [3H]fucose that was isolated from n eu r o b las to ma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation o f the cells, we observed a rapid metabolism (27% by 2 h) o f the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was n o t due to exocytosis since less than 4% of the [3H]fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence o f 40 mM fucose in the media did n o t affect cell growth until after 24 h o f incubation or cellular protein synthesis until after 15 h of incubation. When the metabolism of neurobl ast om a cell glycoproteins was measured in the presence of 1.8 • 10 -4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization o f [3H]fucose. Although the major p r o p o r t i o n of the radioactivity remained as [3H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts o f tritium associated with ot her cellular co mp o n e nt s . However, a rapid rate of glycoprotein metabolism could also be d e m o n s t r a t e d with cells incubated with [14C]fucose. This eliminated the possibility t ha t the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange. Introduction The synthesis and degradation of glycoproteins has been o f interest to investigators in several areas of cell biology [1--12]. Radioactive fucose (6-deoxy-
568 galactose) has been used extensively to study glycoprotein metabolism in a wide variety of mammalian cells [8--12]. Radioisotopic studies with this monosaccharide have proven useful since it is generally thought to be relatively stable and primarily incorporated directly into macromolecules [13,14]. In addition, it has been shown that eukaryotic cells primarily incorporate fucose into plasma membrane components [12,15,16], and that this incorporation largely occurs after the synthesis of the polypeptide-oligosaccharide molecules is almost completed [ 12,16]. Glycoprotein metabolism in murine neuroblastoma cells is of particular interest. These cells possesses many of the biochemical features associated with neurons of the central nervous system [17--19], as well as the maintenance of a bioelectrically active surface membrane [20,21]. Furthermore, various lines of neuroblastoma cells have been shown to undergo a dramatic alteration in morphology depending on the culture conditions [19,22], or by the presence of various compounds which can affect the cell growth rate [17,23] and/or macromolecular metabolism [24,25]. Although the plasma membranes of neuroblastoma cells has been shown to be rich in fucosylated macromolecules [7,26,27], there is little information presently available concerning the turnover of glycoproteins. An earlier report suggested that glycoprotein metabolism in the surface membrane of neuroblastoma cells may be rapid, and that several plasma membrane-bound glycoproreins may be metabolized at differential rates [7]. The present study was undertaken using the N2a line of murine neuroblastoma cells, and was designed to characterize the incorporation and localization of radioactive fucose in cellassociated macromolecules. In addition, we have measured the rate of degradation of fucosylated macromolecules, and determined the proportion of radioactivity that remain associated with fucose during various periods of incubation of the cells with the radioactive precursor. Materials and Methods Materials. All cell culture medium components were purchased from Grand Island Biological Co. (Grand Island, N.Y.), unlabelled L-fucose, cycloheximide and puromycin from Sigma Chemical Co. (St. Louis, Mo.) and radioactive chemicals from New England Nuclear Corp. (Boston, Mass). The L-[~4C]fucose (53.2 Ci/mol) was used at 0.2 pCi/ml (3.7 nmol/ml, L-[3H]fucose (12.1 Ci/ mmol) at 1--4 pCi/ml (0.08--0.30 nmol/ml), [3H]tryptophan (8 Ci/mmol) at 1 pCi per ml and [14C]isoleucine (283 Ci/mol) at 1.0 pCi per ml of complete media. N e u r o b l a s t o m a cell culture. A stock culture of neuroblastoma cells (clone N2a), obtained from Dr. A. Telsar, Northwestern University Medical School, was maintained as monolayer cultures in Falcon tissue culture flasks (75 cm 2) at 37°C, under CO2/air (5 : 95, v/v), in humidified incubators. The cultures were transferred by Viokase treatment and replated in fresh medium which was composed of Dulbecco's Modified Eagle's medium supplemented with dextrose (3.5 g/l), NaHCO3 (3.7 g/l), penicillin (1000 units/I), streptomycin (1000 pg/1), fungizone (2500 pg/1) and 10% 7-globulin-free newborn calf serum. I n c u b a t i o n o f n e u r o b l a s t o m a cells in radioactive precursors. For the experi-
569 ments described, cells were routinely grown and labelled as subconfl uent monolayer cultures in com pl e t e medium. These cultures were transferred 2--3 days before incubation with radioactive precursors, to allow cells sufficient time to recover f r o m the Viokase t r e a t m ent . Neuroblastoma cells were always labelled with [3H]fucose in com pl e t e medium supplemented with 2.5 pM unlabelled L-fucose. This c o n c e n t r a t i o n of unlabelled fucose was shown to be at saturation and was included to avoid the possibility t hat L-fucose was rate limiting t o cell metabolism. In the case of [~4C]fucose unlabelled L-fucose was n o t added since the radioactive precursor was already at a saturating concentration. Cells were incubated in ['4C]isoleucine or [ 3 H ] t r y p t o p h a n in complete medium essentially as described by Mathews et al. [7]. Analysis of glycoprotein turnover in whole cells. Cells were prelabelled in 60mm Falcon tissue culture dishes with L-[3H]fucose (as described a b o v e ) f o r 24 h in order to obtain steady-state labelling. At that time all the cultures were washed twice with fresh medium and reincubated in com pl et e medium containing 40 mM unlabelled L-fucose. After incubation the monolayers were washed twice with ice-cold 0.15 M NaC1, 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH was added to the dish, and the viscous lysate which resulted was transferred to a thick walled test tube. A 0.5 ml aliquot of 10% trichloroacetic acid was then pipetted o n t o the plate, and the solution swirled to remove residual cell material. Both samples were pooled, mixed and the acid-insoluble precipitate was pelleted by centrifugation at 2000 × g for 10 min. The acid-soluble supernatant fluid was assayed for radioactivity where indicated, and the remainder o f the fluid was discarded. The acid-insoluble material in the pellet was resolubilized by the addition of 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH. The solution was mixed, and a 0.2 ml aliquot was removed, and the a m o u n t of radioactivity was measured in White's scintillation fluid [28]. A second 0.2 ml aliquot of the solubilized pellet was removed for the quantitation of protein by the s p e c t r o p h o t o m e t r i c m e t h o d of L o w r y et al. [29]. Extraction of glycolipids. Neuroblastoma cells were assayed for incorporation o f L-[3H]fucose into glycolipids by a modification of the m e t h o d described by Bosmann et al. [10]. Cells were incubated for 24 h with [3H]fucose (as described above), and the macromolecules were precipitated with trichloroacetic acid as previously described. The precipitate was removed by centrifugation at 2000 × g and the pellet was resolubilized with 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH. The cell e xt ract was reprecipitated, centrifuged, and resolubilized three times in order to remove exogenous [3H]fucose. The final pellet was e xt r act ed three successive times with 3 ml of c h l o r o f o r m / m e t h anol (2 : 1, v/v) for 10 min at r o o m t e m p e r a t u r e with vigorous mixing, and then the samples were centrifuged at 3200 × g for 15 min. The c h l o r o f o r m / m e th an o l supernatant fluid was carefully removed and the c h l o r o f o r m / m e t h anol-insoluble pellet was resolubilized in 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH. An aliquot o f the supernatant fluid and the resolubilized pellet was transferred to scintillation vials, and the a m o u n t o f radioactivity in the organic solvent and the c hl or of or m / m et hanol - i nsol ubl e material was determined. Thin-layer chromatography. The pe r cent o f tritium associated with fucose after incubation with neuroblastoma cells was determined by thin-layer chromatography. Cell samples were lyophilized until dry, resuspended in 1 M HCI,
570 sealed in 6 X 50 mm test tubes and heated for 1 h at 100°C. Upon cooling to room temperature the cellular material was lyophilized and resuspended in distilled water. A portion of each hydrolysate was applied in pl quantities to glasssilica gel plates, and run in the butanol/H20/pyridine (6 : 3 : 4, v/v) solvent system until the solvent front reached 60--70% of the height of the plate [30]. Reducing sugar of the standard carbohydrates was visualized by spraying a KMnO4 (0.5 g/15 ml) solution onto the glass plate and circling the developed spots as they appeared [31]. The position of radioactive material was then ascertained by scraping the silica gel from the sample origin to the solvent front in 0.5-cm sections. Results
Before experiments were carried out to determine the characteristics of turnover of fucosylated components, a series of studies was initiated to measure both the kinetics of L-[3H]fucose uptake into acid-soluble pools and the incorporation of the radioactive precursor into acid-insoluble macromolecules. The acid-soluble intracellular pools were found to reach steady-state kinetics within 2 h of incubation (Fig. 1) and steady-state levels were maintained throughout 36 h of incubation. However, the rate of [3H]fucose incorporation into acidinsoluble material was linear for at least 9 h and did not approach steady-state conditions until approx. 12 h of incubation (Fig. 1). In light of these kinetics, in all subsequent experiments where steady-state conditions were necessary, cells were incubated with [3H]fucose for 24 h to avoid possible preferential labelling of glycoproteins. Since glycoproteins and glycolipids are both known to contain fucose, the percentage of radioactive fucose associated with gl~colipids in neuroblastoma cells was measured. Monolayer cultures were prelabelled for 24 h with [3H]fucose, the cells were washed with fresh medium, the macromolecules were precipitated with trichloroacetic acid which were then
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F i g . 1. T h e k i n e t i c s o f [ 3 H ] f u c o s e i n c o r p o r a t i o n i n t o a c i d - s o l u b l e a n d a c i d - i n s o l u b l e f r a c t i o n s o f n e u r o b l a s t o m a cells. M o n o l a y e r s c u l t u r e s w e r e i n c u b a t e d w i t h [ 3 H ] f u e o s e (1 p C i / m l ) i n c o m p l e t e m e d i u m s u p p l e m e n t e d w i t h 2 . 5 p M u n l a b e l l e d L - f u e o s e . A f t e r v a r i o u s p e r i o d s o f i n c u b a t i o n t h e a c i d - s o l u b l e (o) a n d a c i d - i n s o l u b l e ( e ) c e l l u l a r r a d i o a c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s .
571
extracted with chloroform/methanol (2 : 1, v/v). In three independent experiments less then 3% of the radioactivity in the trichloroacetic acid-insoluble material was extracted by chloroform/methanol and it appeared that the majority of the fucose was primarily incorporated into glycoproteins. The extent of glycoprotein turnover in neuroblastoma cell monolayers was measured by incubating the cells for 24 h with [3H]fucose, the radioactive medium was removed, the cells were washed twice with complete medium, and the culture was overlaid with complete medium supplemented with 40 mM unlabelled L-fucose. A 40 mM concentration of unlabelled fucose was included during the chase period in order to limit reutilization of cell-associated [3H]fucose. In a series of preliminary experiments a final concentration of 40 mM non-radioactive fucose was found to reduce the incorporation of [SH]fucose into acid-insoluble macromolecules by over 90%. The prelabelled cells were then reincubated, and at intervals approximating the cell generation time (22 h), the amount of [3H]fucose in trichloroacetic acid-insoluble macromolecules was measured. The loss of radioactivity of the macromolecules was compared to the calculated decrease in cellular specific activity that would be expected if turnover was absent, and dilution of radioactivity occurred solely on the basis of cell growth. The difference between the experimental and the calculated rates represented the measured turnover rate (Fig. 2). In neuroblastoma cells,
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Biochimica et Biophysica Acta, 497 (1977) 567--577 © Elsevier/North-Holland Biomedical Press
BBA 28224 RAPIDLY M E T A B O L I Z E D G L Y C O P R O T E I N S IN A N E U R O B L A S T O M A CELL LINE
JAMES E. HUDSON and TERRY C. JOHNSON Department o f Microbiology-Immunology, The Medical and Dental Schools, Northwestern University, Chicago, Ill. 60611 (U.S.A.)
(Received November 24th, 1976) Summary The metabolism of neuroblastoma cell glycoproteins was examined using L-[3H]fucose. Incubation of m o n o l a y e r cultures with [3H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [3H]fucose that was isolated from n eu r o b las to ma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation o f the cells, we observed a rapid metabolism (27% by 2 h) o f the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was n o t due to exocytosis since less than 4% of the [3H]fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence o f 40 mM fucose in the media did n o t affect cell growth until after 24 h o f incubation or cellular protein synthesis until after 15 h of incubation. When the metabolism of neurobl ast om a cell glycoproteins was measured in the presence of 1.8 • 10 -4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization o f [3H]fucose. Although the major p r o p o r t i o n of the radioactivity remained as [3H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts o f tritium associated with ot her cellular co mp o n e nt s . However, a rapid rate of glycoprotein metabolism could also be d e m o n s t r a t e d with cells incubated with [14C]fucose. This eliminated the possibility t ha t the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange. Introduction The synthesis and degradation of glycoproteins has been o f interest to investigators in several areas of cell biology [1--12]. Radioactive fucose (6-deoxy-
568 galactose) has been used extensively to study glycoprotein metabolism in a wide variety of mammalian cells [8--12]. Radioisotopic studies with this monosaccharide have proven useful since it is generally thought to be relatively stable and primarily incorporated directly into macromolecules [13,14]. In addition, it has been shown that eukaryotic cells primarily incorporate fucose into plasma membrane components [12,15,16], and that this incorporation largely occurs after the synthesis of the polypeptide-oligosaccharide molecules is almost completed [ 12,16]. Glycoprotein metabolism in murine neuroblastoma cells is of particular interest. These cells possesses many of the biochemical features associated with neurons of the central nervous system [17--19], as well as the maintenance of a bioelectrically active surface membrane [20,21]. Furthermore, various lines of neuroblastoma cells have been shown to undergo a dramatic alteration in morphology depending on the culture conditions [19,22], or by the presence of various compounds which can affect the cell growth rate [17,23] and/or macromolecular metabolism [24,25]. Although the plasma membranes of neuroblastoma cells has been shown to be rich in fucosylated macromolecules [7,26,27], there is little information presently available concerning the turnover of glycoproteins. An earlier report suggested that glycoprotein metabolism in the surface membrane of neuroblastoma cells may be rapid, and that several plasma membrane-bound glycoproreins may be metabolized at differential rates [7]. The present study was undertaken using the N2a line of murine neuroblastoma cells, and was designed to characterize the incorporation and localization of radioactive fucose in cellassociated macromolecules. In addition, we have measured the rate of degradation of fucosylated macromolecules, and determined the proportion of radioactivity that remain associated with fucose during various periods of incubation of the cells with the radioactive precursor. Materials and Methods Materials. All cell culture medium components were purchased from Grand Island Biological Co. (Grand Island, N.Y.), unlabelled L-fucose, cycloheximide and puromycin from Sigma Chemical Co. (St. Louis, Mo.) and radioactive chemicals from New England Nuclear Corp. (Boston, Mass). The L-[~4C]fucose (53.2 Ci/mol) was used at 0.2 pCi/ml (3.7 nmol/ml, L-[3H]fucose (12.1 Ci/ mmol) at 1--4 pCi/ml (0.08--0.30 nmol/ml), [3H]tryptophan (8 Ci/mmol) at 1 pCi per ml and [14C]isoleucine (283 Ci/mol) at 1.0 pCi per ml of complete media. N e u r o b l a s t o m a cell culture. A stock culture of neuroblastoma cells (clone N2a), obtained from Dr. A. Telsar, Northwestern University Medical School, was maintained as monolayer cultures in Falcon tissue culture flasks (75 cm 2) at 37°C, under CO2/air (5 : 95, v/v), in humidified incubators. The cultures were transferred by Viokase treatment and replated in fresh medium which was composed of Dulbecco's Modified Eagle's medium supplemented with dextrose (3.5 g/l), NaHCO3 (3.7 g/l), penicillin (1000 units/I), streptomycin (1000 pg/1), fungizone (2500 pg/1) and 10% 7-globulin-free newborn calf serum. I n c u b a t i o n o f n e u r o b l a s t o m a cells in radioactive precursors. For the experi-
569 ments described, cells were routinely grown and labelled as subconfl uent monolayer cultures in com pl e t e medium. These cultures were transferred 2--3 days before incubation with radioactive precursors, to allow cells sufficient time to recover f r o m the Viokase t r e a t m ent . Neuroblastoma cells were always labelled with [3H]fucose in com pl e t e medium supplemented with 2.5 pM unlabelled L-fucose. This c o n c e n t r a t i o n of unlabelled fucose was shown to be at saturation and was included to avoid the possibility t hat L-fucose was rate limiting t o cell metabolism. In the case of [~4C]fucose unlabelled L-fucose was n o t added since the radioactive precursor was already at a saturating concentration. Cells were incubated in ['4C]isoleucine or [ 3 H ] t r y p t o p h a n in complete medium essentially as described by Mathews et al. [7]. Analysis of glycoprotein turnover in whole cells. Cells were prelabelled in 60mm Falcon tissue culture dishes with L-[3H]fucose (as described a b o v e ) f o r 24 h in order to obtain steady-state labelling. At that time all the cultures were washed twice with fresh medium and reincubated in com pl et e medium containing 40 mM unlabelled L-fucose. After incubation the monolayers were washed twice with ice-cold 0.15 M NaC1, 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH was added to the dish, and the viscous lysate which resulted was transferred to a thick walled test tube. A 0.5 ml aliquot of 10% trichloroacetic acid was then pipetted o n t o the plate, and the solution swirled to remove residual cell material. Both samples were pooled, mixed and the acid-insoluble precipitate was pelleted by centrifugation at 2000 × g for 10 min. The acid-soluble supernatant fluid was assayed for radioactivity where indicated, and the remainder o f the fluid was discarded. The acid-insoluble material in the pellet was resolubilized by the addition of 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH. The solution was mixed, and a 0.2 ml aliquot was removed, and the a m o u n t of radioactivity was measured in White's scintillation fluid [28]. A second 0.2 ml aliquot of the solubilized pellet was removed for the quantitation of protein by the s p e c t r o p h o t o m e t r i c m e t h o d of L o w r y et al. [29]. Extraction of glycolipids. Neuroblastoma cells were assayed for incorporation o f L-[3H]fucose into glycolipids by a modification of the m e t h o d described by Bosmann et al. [10]. Cells were incubated for 24 h with [3H]fucose (as described above), and the macromolecules were precipitated with trichloroacetic acid as previously described. The precipitate was removed by centrifugation at 2000 × g and the pellet was resolubilized with 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH. The cell e xt ract was reprecipitated, centrifuged, and resolubilized three times in order to remove exogenous [3H]fucose. The final pellet was e xt r act ed three successive times with 3 ml of c h l o r o f o r m / m e t h anol (2 : 1, v/v) for 10 min at r o o m t e m p e r a t u r e with vigorous mixing, and then the samples were centrifuged at 3200 × g for 15 min. The c h l o r o f o r m / m e th an o l supernatant fluid was carefully removed and the c h l o r o f o r m / m e t h anol-insoluble pellet was resolubilized in 0.5 ml of 0.15 M NaC1 containing 0.2 M NaOH. An aliquot o f the supernatant fluid and the resolubilized pellet was transferred to scintillation vials, and the a m o u n t o f radioactivity in the organic solvent and the c hl or of or m / m et hanol - i nsol ubl e material was determined. Thin-layer chromatography. The pe r cent o f tritium associated with fucose after incubation with neuroblastoma cells was determined by thin-layer chromatography. Cell samples were lyophilized until dry, resuspended in 1 M HCI,
570 sealed in 6 X 50 mm test tubes and heated for 1 h at 100°C. Upon cooling to room temperature the cellular material was lyophilized and resuspended in distilled water. A portion of each hydrolysate was applied in pl quantities to glasssilica gel plates, and run in the butanol/H20/pyridine (6 : 3 : 4, v/v) solvent system until the solvent front reached 60--70% of the height of the plate [30]. Reducing sugar of the standard carbohydrates was visualized by spraying a KMnO4 (0.5 g/15 ml) solution onto the glass plate and circling the developed spots as they appeared [31]. The position of radioactive material was then ascertained by scraping the silica gel from the sample origin to the solvent front in 0.5-cm sections. Results
Before experiments were carried out to determine the characteristics of turnover of fucosylated components, a series of studies was initiated to measure both the kinetics of L-[3H]fucose uptake into acid-soluble pools and the incorporation of the radioactive precursor into acid-insoluble macromolecules. The acid-soluble intracellular pools were found to reach steady-state kinetics within 2 h of incubation (Fig. 1) and steady-state levels were maintained throughout 36 h of incubation. However, the rate of [3H]fucose incorporation into acidinsoluble material was linear for at least 9 h and did not approach steady-state conditions until approx. 12 h of incubation (Fig. 1). In light of these kinetics, in all subsequent experiments where steady-state conditions were necessary, cells were incubated with [3H]fucose for 24 h to avoid possible preferential labelling of glycoproteins. Since glycoproteins and glycolipids are both known to contain fucose, the percentage of radioactive fucose associated with gl~colipids in neuroblastoma cells was measured. Monolayer cultures were prelabelled for 24 h with [3H]fucose, the cells were washed with fresh medium, the macromolecules were precipitated with trichloroacetic acid which were then
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F i g . 1. T h e k i n e t i c s o f [ 3 H ] f u c o s e i n c o r p o r a t i o n i n t o a c i d - s o l u b l e a n d a c i d - i n s o l u b l e f r a c t i o n s o f n e u r o b l a s t o m a cells. M o n o l a y e r s c u l t u r e s w e r e i n c u b a t e d w i t h [ 3 H ] f u e o s e (1 p C i / m l ) i n c o m p l e t e m e d i u m s u p p l e m e n t e d w i t h 2 . 5 p M u n l a b e l l e d L - f u e o s e . A f t e r v a r i o u s p e r i o d s o f i n c u b a t i o n t h e a c i d - s o l u b l e (o) a n d a c i d - i n s o l u b l e ( e ) c e l l u l a r r a d i o a c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s .
571
extracted with chloroform/methanol (2 : 1, v/v). In three independent experiments less then 3% of the radioactivity in the trichloroacetic acid-insoluble material was extracted by chloroform/methanol and it appeared that the majority of the fucose was primarily incorporated into glycoproteins. The extent of glycoprotein turnover in neuroblastoma cell monolayers was measured by incubating the cells for 24 h with [3H]fucose, the radioactive medium was removed, the cells were washed twice with complete medium, and the culture was overlaid with complete medium supplemented with 40 mM unlabelled L-fucose. A 40 mM concentration of unlabelled fucose was included during the chase period in order to limit reutilization of cell-associated [3H]fucose. In a series of preliminary experiments a final concentration of 40 mM non-radioactive fucose was found to reduce the incorporation of [SH]fucose into acid-insoluble macromolecules by over 90%. The prelabelled cells were then reincubated, and at intervals approximating the cell generation time (22 h), the amount of [3H]fucose in trichloroacetic acid-insoluble macromolecules was measured. The loss of radioactivity of the macromolecules was compared to the calculated decrease in cellular specific activity that would be expected if turnover was absent, and dilution of radioactivity occurred solely on the basis of cell growth. The difference between the experimental and the calculated rates represented the measured turnover rate (Fig. 2). In neuroblastoma cells,
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