Radioactive Glycoproteins Research Articles

N-Glycosylation of proteins by homogenate preparations of developing chick embryos.

Homogenate preparations of chick embryos contain the enzymes and endogenous precursors required for protein N-glycosylation mediated by dolichyl phosphate. The rate and extent of protein N-glycosylation was relatively high. Sensitivity of the first step in the dolichyl phosphate cycle to tunicamycin was confirmed. That tunicamycin had no indirect effect on the incorporation of glucose into lipid-linked intermediates or into protein but did reduce mannose incorporation was interpreted as supporting the view that the final glucosylation of the oligosaccharide is rate-limiting. Endogenous nucleotide diphosphate sugars and sugar acceptors, both lipid and protein linked, were present in the membrane preparation. Studies using dodecylsulphate-polyacrylamide gel electrophoresis on the radioactive glycoproteins formed during the incubations with nucleoside diphosphate [14C]sugars indicated that approximately one third of the 43 proteins detected carried radioactivity. Two thirds of the glycoproteins formed carried radioactive oligosaccharide chains that were either fully glucosylated or had been subsequently trimmed and received peripheral residues of N-acetylglucosamine. Most of the glycoproteins also carried radioactive oligosaccharide chains at varying stages of glycosylation preceding glucosylation and some carried only these ‘immature7rsquo; oligosaccharides. The transfer of mannose to protein and lipids was most active at 7 or 9 days of development being less active in both younger and older embryos whereas the pattern of change of activities of transfer of glucose and N-acetylglucosamine was more complicated. Most of the radioactive glycoproteins prominent at day 9 of development were also prominent in younger or older embryos. However, the incorporation of radioactive sugars into some glycoproteins, relative to that incorporated generally was very dependent upon the age of the embryos used.

Mucin exocytosis: a major target for Helicobacter pylori.

To determine whether Helicobacter pylori impairs the secretory function of mucous cells. The mucus secreting human cell line CL. 16E, maintained as confluent monolayers on nitrocellulose filters, was infected with H pylori strain CIP 101260. After three hours of incubation with H pylori the monolayers were washed and reincubated with fresh culture medium for various time periods (24, 48, or 72 hours) before evaluating both the morphology and function of mucous cells. For morphological studies, epithelial monolayers were fixed in situ and processed for both standard histochemistry on paraffin wax sections, and electron microscopy. To measure mucins secreted from cultured cells, the cells were metabolically labelled with 3H-glucosamine. Undegraded mucins were quantitated as the radioactive glycoproteins blocked at the stacker gel interface after sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the secretory glycoproteins. Control cultures of CL. 16E cells grew on filters as homogeneous monolayers of polarised mucous cells secreting a visco-elastic gel of mucins at the apical surface. In infected monolayers H pylori was in close contact with the apical surface of mucous cells. Cell counts and histological evaluation of the monolayers did not reveal any significant deleterious effect of H pylori on the mucous cells. H pylori induced only a modest inhibition of baseline mucus secretion from CL. 16E cells, this inhibition being significant only at 24 hours. In contrast, the mucus secretory response to two agents that raise intracellular cAMP and calcium--forskolin and ionophore A23187--was strongly inhibited. The inhibitory effect of H pylori on the exocytotic response was not paralleled by an inhibition of glycoprotein synthesis. Considering the fact that the exocytotic response to a variety of secretagogues constitutes the primary line of defence of the gastric mucosa in an emergency, it is suggested that H pylori exerts its deleterious effects by weakening this important physiological defence.

Oligosaccharide composition of the neurotoxin-responsive sodium channel of mouse neuroblastoma and requirement of sialic acid for biological activity

A glycoprotein, M r, 200000, which has the biological activity of the neurotoxin-responsive Na + channel, was isolated from a clonal line of mouse neuroblastoma cells, N-18. The glycoprotein was purified to homogeneity in 18% yield by methods used to purify glycoproteins, which included metabolic labeling of the cells with l-[ 3H]fucose and binding of the radioactive glycoproteins to WGA- and lentil-Sepharose, and DEAE-cellulose. The glycoprotein has biological activity of neurotoxin-responsive ion flux when reconstituted into artificial phospholipid vesicles. This activity was shown to depend on the presence of sialic acid since treatment of the purified, reconstituted glycoprotein with Vibrio cholerae neuraminidase abolished the response to neurotoxins of 86Rb flux. The [ 3H]fucose-containing glycopeptides derived by Pronase digestion of the glycoprotein were characterized by affinity to immobilized lectins and contained di-, tri-, and tetra-antennary oligosaccharides in a ratio of 2:4:3. Most of the glycopeptides were sialylated as shown by binding characteristics to immobilized serotonin-Sepharose with and without neuraminidase. The structure of the diantennary oligosaccharides was elucidated by 500-MHz 1H NMR spectroscopy. The Con A-bound fraction contains α-NeuNAc-(2 → 6)-bound group on the GlcNAc 5′ antenna and an α-NeuNAc-(2 → 3)-bound groups on the GlcNAc 5 antenna. An α- l-fucosyl group is (1 → 6)-bound to the Asn core GlcNAc 1 residue.

Sulphation causes heterogeneity of gastric mucins

The synthesis of mucus glycoprotein in rat stomach was studied in stomach segments, which were pulse-labelled with both [3H]galactose and [35S]sulphate and chased for various times. The radioactive glycoproteins were analyzed by CsCl centrifugation and by agarose gel electrophoresis. After a pulse-labelling for 15 min with [3H]galactose, a possible intermediate with an Mr of 200,000 and a buoyant density of 1.60 g/ml could be demonstrated. Following chase periods of 1 and 4 h, [3H]galactose and [35S]sulphate were present in glycoproteins with a mean buoyant density of 1.50 g/ml. This is clearly different from the main density of glycoproteins isolated from mucosal scrapings (1.46 g/ml). Another difference is the high electrophoretic mobility on gel electrophoretic analysis of newly synthesized glycoproteins compared to that of the major portion of the glycoproteins from mucosal scrapings. When sulphation of glycoproteins was inhibited by sodium chlorate, electrophoretic mobility and buoyant density both decreased. Sodium chlorate had no effect on glycoprotein synthesis nor on glycoprotein secretion. We conclude from our data that the heterogeneity in electrophoretic mobility and buoyant density can be attributed to a different degree of sulphation of the same glycoprotein.

Characteristics of two wheat germ agglutinin-resistant variants of B16 mouse melanoma cells with reduced tumorigenicity

Two variants of B16 mouse melanoma cells, selected for their resistance to toxic levels of wheat germ agglutinin isolectin 1 (WGA-1) in serum-free medium, showed by chromosome analysis that they are still mouse cell-lines, continue to produce melanin, and are less tumorigenic in mice than the parent B16 cells. The variants showed a marked decrease in cell agglutination with the wheat germ lectin and a slight increse in cell agglutination with concanavalin A. The binding of 125I-labeled wheat germ agglutinin to the two variant lines was likewise decreased over a 10 3-fold range of lectin concentrations. Terminal sialyl residues were critical in WGA-1 binding to the wild-type cells. The binding data indicated a decrease in high-affinity binding as well as a decrease in the total number of binding sites in the variants. Polyacrylamide gel electrophoresis, followed by affinity staining with 125I-wheat germ agglutinin, showed alterations in the what germ agglutinin-binding glycoproteins in the variants compared to those of the parent cell line. However, lactoperoxidase-catalyzed iodination revealed a similar cell-surface protein pattern among the three cell lines. Radioactive glycoproteins secreted or shed by the three cell lines grown in the presence of [ 3H]glucosamine in serum-free medium were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/mL). The WGA-bound glycoproteins from the two variants had molecular weights of 92,000, 56,000, and 42,000. None of these components was detected in the parent cell-line. A major WGA-binding glycoprotein, which accounted for 37% of the total [ 3H]glucosamine incorporated, was isolated from the spent medium of the parent mouse melanoma cell-line. This glycoprotein was apparently absent in the WGA-1-resistant variants.

Biosynthesis, processing and secretion of mucus glycoprotein in the rat stomach

For the study of the biosynthesis, processing and secretion of mucus glycoproteins in rat gastric mucous cells, antibodies were raised against purified gastric mucus glycoproteins and against deglycosylated gastric mucus glycoproteins. Indirect immunofluorescence analysis of gastric mucosa sections revealed that both antibodies specifically labelled the mucus glycoprotein-synthesizing cells in the gastric mucosa. Stomach segments were pulse-labelled with [ 35S]cysteine and chased for various times. The radioactively labelled (glyco)proteins were quantitatively immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. Less than 3% of the total radioactivity incorporated in protein was found to be present in mucus glycoproteins. Antibodies raised against native mucus glycoproteins recognized only high-molecular-weight mucus glycoproteins, while the antibodies against deglycosylated glycoproteins also bound to probable precursor forms. The synthesis of mature mucus glycoproteins ( M r > 300 000) required about 90 min. After 3 h of chase, only a small portion of the pulse-labelled mucus glycoproteins had been secreted; the majority of the radioactive glycoproteins at that time was still associated with the tissue. Immature (glyco)proteins were not secreted into the medium.

Colonic glycoprotein secretion and calmodulin-acceptor proteins in the reserpine-treated rat.

The rate of radioactive precursor-labeled colonic glycoprotein secretion in chronically reserpine-treated versus saline-injected control rats was examined. Everted colonic sacs prepared from reserpine-treated rats were found to be hypersecretory, exhibiting a basal rate of glycoprotein secretion that was threefold higher than control everted sacs. Furthermore, glycoprotein secretion in control tissue was stimulated by the secretagogue carbachol, and this stimulation was precluded by 10 microM trifluoperazine, the calmodulin antagonist. Reserpine-treated tissue, in contrast, was refractory to treatment with carbachol as well as trifluoperazine. While reserpine-treated and control colonic mucosae were demonstrated to contain equivalent levels of calmodulin via radioimmunoassay, reserpine-treated tissue was determined to lack two calmodulin-acceptor proteins with molecular weights of 29 and 47 kilodaltons. The data suggest that the mechanism by which reserpine elicits this cystic fibrosislike, hypersecretory state of glycoprotein secretion within the colonic mucosa entails the loss of calmodulin function in the regulation of this secretory process. We speculate that the biochemical defect present in cystic fibrosis could also entail such a loss of calmodulin function in the regulation of glycoprotein secretion.

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments.

We have examined the rate of dissociation of internalized 125I-asialo-orosomucoid-receptor complexes in freshly isolated rat hepatocytes. Cell suspensions were washed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 0 degrees C to remove surface-bound ligand and then assessed for the retention of radioactive glycoprotein in the presence of digitonin, which permeabilized the cells and released the internal soluble contents. In cells which initially contained only surface-bound ligand, about 50% of the internalized ligand dissociated from receptor very rapidly (t1/2 less than or equal to 2.5 min, k greater than or equal to 0.28 min-1), at 37 degrees C, whereas the other 50% dissociated more slowly with apparent first order kinetics (t1/2 = 50 min, k = 0.014 min-1). This equal distribution of internalized ligand into two compartments, from which dissociation occurred with very different kinetics, did not depend on the extent of surface receptor occupancy and also occurred under non-steady state conditions of continuous exposure to ligand. Ligand entering both the rapid and slow dissociation compartments was eventually degraded with apparent first order kinetics (k = 0.0047 min-1), suggesting that the intracellular routing of ligand to lysosomes after dissociation from either compartment was via the same pathway. The fast and slow dissociation of receptor-ligand complexes were also distinguished by different temperature sensitivities; the slow dissociation process ceased below 18 degrees C, whereas the fast dissociation process still proceeded. The equal partition of internalized complexes into the two kinetic compartments did not change as a function of temperature but did change as cells continued to endocytose asialo-orosomucoid at 37 degrees C. As the internal receptor pool approached a steady state level of occupancy, there was an increase in the average time for receptor recycling and an increase in the fraction of incoming receptor-ligand complexes which dissociated rapidly (approximately 75%). In addition, under steady state conditions, the rate of the slow dissociation process increased (k = 0.026 min-1, t1/2 = 27 min).

Reconstitution in vitro of neurotoxin-responsive ion efflux by using membrane glycoproteins of neuroblastoma cells.

Glycoproteins were purified from a clonal cell line of mouse neuroblastoma, N-18, labeled metabolically with L-[3H]fucose. The purified radioactive glycoproteins were reconstituted into artificial phosphatidylcholine vesicles. When the vesicles were preloaded with cesium acetate and treated with neurotoxins to activate the Na+ channel, a shift in intravesicular density was observed to a less dense position after centrifugation on sucrose gradients. This shift was partially inhibited by tetrodotoxin, which prevents the activation of the Na+ channel. A similarly derived fraction of [14C]fucose-containing glycoproteins from a neuroblastoma cell line that does not possess excitable membranes, N1A-103, was reconstituted into phospholipid vesicles, and, after preloading with cesium ions, the fraction was combined with those of the 3H-labeled glycoproteins of the differentiated cells, N-18, which have excitable membranes. Only the 3H-labeled glycoprotein-containing vesicles were responsive to the neurotoxins, as shown by a shift in intravesicular density on sucrose gradients. These results are interpreted as a demonstration of the reconstitution of glycoproteins to form the activated Na+ channel. Comparison of the radioactive glycoprotein profiles after polyacrylamide gel electrophoresis showed that glycoproteins of Mr 200,000, Mr 165,000, and Mr 65,000 were common to the reconstituted fractions that were biologically active.

Degradation of [6-3H]- and [1-14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver.

We studied the degradation and metabolism of radioactive glucosamine-labeled asialo-alpha 1-acid glycoprotein by the perfused rat liver. Removal of 130 micrograms of the protein from the perfusate occurred with a T 1/2 of 10.3 min. Radioactivity associated initially with the microsomal fraction of the tissue homogenate and was then transferred to the lysosomes where hydrolysis of N-acetylglucosamine residues took place. Radioactivity left the lysosomal-rich fraction of the homogenate with a T 1/2 of 50 min. Final accumulation of radioactivity was in the supernatant and greater than 80% of this material was found to be UDP-N-acetylhexosamines. In those studies, 10 mumol of unlabeled glucosamine had been added to the perfusate to expand the intracellular pool of UDP-GlcNAc and prevent further metabolism of any radioactive N-acetylglucosamine released by the lysosomes. When this procedure was not done, greater than one-third of the lysosomally derived N-acetylglucosamine was reused by the liver to form new radioactive glycoproteins secreted into the perfusate. Overall hydrolysis of N-acetylglucosamine from the glycoprotein substrate was greater than 80% during 2 h of perfusion. However, leupeptin, a thiol cathepsin inhibitor, decreased the release of amino sugar by 50%, even though this compound had no effect on the activity of any lysosomal glycosidase in vitro. Large glycopeptides of molecular weight 35,000 accumulated in the lysosomes due to leupeptin. From these studies, we conclude that efficient digestion of the carbohydrate component of this glycoprotein in situ requires the concerted activity of both lysosomal glycosidases and proteases.

Influence of incubation conditions, tunicamycin and castration on incorporation of [3H]mannose and [3H]fucose into rat epididymal glycoproteins in vitro.

The incorporation of radioactive mannose and fucose into secretory glycoproteins by rat epididymal tissue was studied using tissue pieces in vitro. The appearance of radioactive macromolecular products in the medium occurred after a lag phase of 2 h with radioactive mannose, but with radioactive fucose the lag phase was only 15 min. Preincubation of tissue for 2 h before the addition of radioactive mannose increased the subsequent rate of incorporation by reducing the lag phase from 2 to 1 h. Tunicamycin reduced the incorporation of radioactive mannose and fucose into macromolecular products to approximately 15 and 50% of normal in the caput and cauda respectively; maximum inhibition required 10 micrograms tunicamycin/ml in the caput and 2 micrograms/ml in the cauda. Reduction of radioactive sugar incorporation by tunicamycin did not result in qualitative changes in the profile of the radioactive glycoproteins that were secreted. However, immunoprecipitation of proteins D and E from incubations with radioactive methionine or mannose revealed that tunicamycin caused these proteins to be synthesized and secreted in a non-glycosylated form. Prior castration of animals reduced the incorporation of radioactive mannose and fucose, and qualitative changes in the profiles of secreted radioactive glycoproteins were apparent.

Glycoprotein Synthesis of Renal Collecting Duct Epithelium Cultured as Globular Bodies

Cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's minimum essential medium (DMEM) containing 10% fetal calf serum. Within 24 h the explants formed 'globular bodies' which were completely covered by a monolayered epithelium. The cells were differentiated and resembled collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with D-valine instead of L-valine, a monolayer of collecting duct cells was obtained and used for control experiments. For analysis and identification of synthesized glycoproteins, the globular bodies were incubated with various labelled carbohydrates and amino acids, and then fractionated. Glycoproteins secreted into the culture medium were not detected. Cell-associated glycoproteins were found in crude membrane fractions and then extracted with Triton X-100 for column chromatography, SDS-polyacrylamide electrophoresis in 6 M urea, isoelectrofocusing, and two-dimensional electrophoresis. Two prominent glycoproteins containing galactose and glucosamine were synthesized during the spreading of the epithelium, with an apparent molecular weight of 150,000 and 85,000 (SDS-PAGE). The synthesized glycoproteins differ in their content of radioactive glycoprotein precursor and leucine. The 85,000 dalton monomer glycoprotein has an isoelectric point of 3.5 and was identified by two-dimensional electrophoresis.

Radioimmunoassay of rat submandibular salivary gland mucin

A solid phase radioimmunoassay (RIA) for rat submandibular mucin (RSM) was developed and applied to studies of mucin concentration, antigenicity and secretion. The assay utilizes the affinity of 125I-Protein A for IgG to quantitate antigendashantibody immunocomplexes immobilized in the wells of plastic microtitre plates. The technique was highly reproducible and capable of detecting as little as 3 ng mucin protein. The submandibular glands of rats weighing 150–180 g contained 257 μg of mucin (dry wt) per mg protein, which is equivalent to about 3.6 mg of mucin per gland. The antiserum to RSM was cross-reactive with mucins from rat sublingual and pig submandibular glands, and rat, human and pig small intestine. No cross-reactivity was detected with mucins from mouse, canine or bovine submandibular glands, and there was no evidence that ABH blood-group sugars contributed to mucin antigenicity. The RIA was used to estimate secretion from dispersed rat submandibular gland cells and gave a more specific and accurate assay of mucin release than previous assays of precursor-labelled radioactive glycoproteins in the culture medium. The β-adrenergic agonist, isoproterenol, stimulated immunoreactive mucin secretion from cultured cells to approximately twice the level of unstimulated or propranolol-inhibited controls. The RIA appears to offer promising new approaches for studies on mucin metabolism and secretion in health and disease.

The Effects of the State Of Differentiation of Labeling of Epidermal Cell Surface Glycoproteins

Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with 125I-lectins, and by the metabolic incorporation of L-(3H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells (and also for neoplastic cells) but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with 125I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells.

Use of radioactive glucosamine in the perfused rat liver to prepare alpha 1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues.

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.

Axonal transport, deposition, and metabolic turnover of glycoproteins in the rat optic pathway.

Abstract: Following intraocular injection of [3H]fucose in the rat, radioactive glycoproteins are rapidly transported to the nerve terminals in at least two waves, one with a peak at 8 h and a second with a peak at about a week. The molecular weight distribution of radioactive peptides in ach transport wave as determined by gel electrophoresis in buffers containing sodium dodecyl sulfate is very similar. Most of the many glycopeptides in the first wave of rapid transport pass through the optic tract in unison (apparent half‐life of about 15 h) and are preferentially destined for the nerve endings. However, two proteins of apparent M. W. 28,000 and 49,000 are preferentially retained in the axons. The remaining proteins, after reaching the nerve endings (superior colliculus), decay with apparent half‐lives ranging from 17 to 34 h. During the second wave a large amount of the 28,000 and 49,000 M. W. peptides are again preferentially retained in the axons. The remaining proteins, on reaching the nerve endings, decay with apparent half‐lives ranging from 5 to 9 days. Subcellular fractionation of the superior colliculus supports the hypothesis that the 49,000 and 28,000 M. W. peptides are the predominantly labeled glycoproteins present in myelinated axons (representing over 50% of the radioactive glycoproteins 7 days following injection), although they are probably also present in membranes of the nerve endings. A comparison with glycoprotein transport in other tracts (geniculocortical and nigrostriatal tracts) suggests that glycoprotein transport in these pathways has many similarities to glycoprotein transport in the retinal ganglion cells, and that the optic system is a good general model for axonal transport in the CNS.

Mannosyl transferases inSaccharomyces cerevisiae: Evidence for the occurrence of ectomannosyltransferase activity

The subcellular distribution of mannosyltransferases inSaccharomyces cerevisiae was studied following the separation of the plasma membrane from other intracellular membranous systems. Most of the activity was linked to internal membranes, and the rest was located at the level of the plasma membrane. Yeast plasma membranes coated on their external face with concanavalin A when incubated with GDP-[U-14C]mannose incorporated 20% less [U-14C]mannose in glycoproteins and 110% more in glycolipids than plasma membranes alone. This suggested that part of the total mannosyltransferase activity of the plasma membrane is located on its outer surface. A significant incorporation of radioactive mannose into trichloroacetic-acid-precipitable material was detected in incubations of protoplasts with GDP-[U-14C]mannose when incorporation of free mannose did not occur. Characterization of a product synthesized by the ectotransferase(s) was achieved after treatment of the radioactive plasma membranes by Triton X-100, which preserved the concanavalin A-mannoprotein complexes and removed a large amount of other plasma membrane components. A radioactive glycoprotein band with an apparent molecular weight of 94, 000 was identified as a product of the ectomannosyltransferase(s).

Glycosylation of myelin glycoproteins in peripheral nerve via lipid intermediates.

Membrane preparations from chick peripheral nervous system (PNS) catalyzed the transfer of [3H]glucose from UDP-[3H]glucose into glucosylphosphoryl dolichol. The initial rate of glucosylphophoryl dolichol formation in a non-myelin membrane fraction from actively myelinating chick PNS was 11 fold higher than that from adult. Exogenous dolichyl monophosphate stimulated glucosylphosphoryl dolichol synthesis in both fractions. The higher level of glucosylphosphoryl dolichol synthesis corresponded to the onset of myelination in chick PNS. Exogenous dolichyl monophosphate also stimulated the labeling of glucosylated oligosaccharide lipids and glycoproteins in the fraction. On SDS polyacrylamide gel electrophoresis, the relative mobility of the major and minor radioactive glycoprotein corresponded with that of the P0 and PASII glycoprotein in PNS myelin, respectively. The results suggest that myelin glycoproteins in PNS are glycosylated via lipid intermediates.

755 GLYCOPROTEINS IN CULTURE MEDIUM: A COMPARISON FROM CYSTIC FIBROSIS (CF) AND CONTROL SKIN FIBROBLASTS

Glycoproteins of high molecular weight are found associated with the cell surface or in growth media of fibroblasts and other cell types in culture. Several abnormalities of glycoproteins in CF have been reported. Therefore the glycoproteins from the media of CF and matched control skin fibroblasts were isolated and partially characterized. Cells were labeled with L-[3H]fucose and grown in medium containing 10% serum. For some experiments the cells were subsequently grown in serum free medium for 48 hours prior to harvest. Media were examined from cells in both log and confluent phases of the growth cycle. The four methods used to isolate the glycoproteins were cryoprecipitation, ammonium sulfate precipitation, heparin precipitation and a heparin-sepharose column. The precipitated proteins were characterized by polyacrylamide gel electrophoresis, isoelectric focusing and protein and carbohydrate content. Different classes of glycoproteins were obtained with each of the four methods. Only heparin precipitation yielded a single radioactive glycoprotein (Mr>200,000) demonstrated by gel electrophoresis. Several qualitative and quantitative differences were noted. In all cases less protein per cell was precipitated by heparin from the CF medium when compared to control. USPHS grant AM16859 and CF Foundation Student Traineeships.

Specific monosaccharide inhibition of active sodium channels in neuroblastoma cells.

L-Fucose and D-galactose in low concentrations (0.27 or 2.7 mM) inhibited the induction of active Na+ channels in mouse and human neuroblastoma cells when the monosaccharides were added to the culture medium for 4 days with the inducing agent dimethyl sulfoxide. Active Na+ ionophores were determined by measurement of the toxin-stimulated efflux of 86Rb from the cells. At the same time, the amount of a radioactive glycoprotein (Mr 200,000), which was shown previously to be associated with neurite and membrane preparations from cells with active Na+ channels, was decreased. Cell growth and viability were not affected. The nonphysiological isomer D-fucose or the addition of D-glucose in the same concentration did not inhibit differentiation. Vibrio cholerae neuraminidase, added to the cells prior to the stimulation of 86Rb efflux by veratridine and scorpion venom, was inhibitory. The implications of these findings, which suggest a key role for glycoproteins in at least a portion of the excitability process, are discussed.