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PubMed1985

Effects of tunicamycin on the expression of beta-adrenergic receptors in human astrocytoma cells during growth and recovery from agonist-induced down-regulation.

Tunicamycin, which inhibits formation of asparagine-linked glycoproteins, caused a concentration-dependent blockade of beta-adrenergic receptor (beta-AR) accumulation in 1321N1 human astrocytoma cells during growth in culture. A concentration of tunicamycin (0.1 microgram/ml) that inhibited receptor accumulation and [3H]mannose or [3H]glucosamine incorporation into glycoproteins by 90% had only a small effect (10%) on [3H]leucine incorporation into protein, and reduced the rate of cell growth. Incubation in drug-free medium subsequent to treatment of 1321N1 cells with tunicamycin for 48 hr resulted in recovery of beta-AR to control levels within an additional 48 hr. Exposure of cultures to isoproterenol (0.1 microM, 12 hr) caused an 80-90% loss of beta-AR in both pre- and postconfluent cultures; beta-AR recovered to control levels upon removal of isoproterenol. Although both tunicamycin and the protein synthesis inhibitor cycloheximide blocked beta-AR accumulation during growth of 1321N1 cells, neither agent inhibited the appearance of beta-AR during recovery from the down-regulated state in preconfluent cultures. However, cycloheximide, but not tunicamycin, blocked recovery of beta-AR after isoproterenol-induced loss of receptors in postconfluent cultures. In a previous report (Mol. Pharmacol. 26:424-429, 1984), we provided direct evidence that recovery of beta-AR from down-regulation in postconfluent cultures requires de novo synthesis of receptor protein. Thus, the results with tunicamycin are consistent with the idea that recovery of beta-AR in postconfluent cultures requires the synthesis of new beta-AR molecules, but as aglycoproteins that exhibit radioligand-binding characteristics similar to those of native glycoprotein beta-AR.

Astrocytoma↗

PubMed1986

Effect of microsomal enzyme inducers and inhibitors on the toxicity of tunicamycin and corynetoxin, the causal agent of annual ryegrass toxicity.

Of the microsomal enzyme inducers and inhibitors tested in rats, the only one to modify the toxicity of tunicamycin or the closely-related corynetoxin, the causal agent of annual ryegrass toxicity, was phenobarbitone. It increased the LD50 of tunicamycin from 0.31 mg/kg to 0.77 mg/kg. Similar results were obtained with the related corynetoxin. Tunicamycin was also shown to be an inhibitor of cytochromes P-450. Rats sacrificed 48 h after receiving an LD50 dose of tunicamycin had cytochromes P-450 levels 30% that of controls. Rats pretreated with trans-stilbene oxide, which like phenobarbitone is an inducer of glutathione transferase, showed no decreased susceptibility to tunicamycin. These results suggest that neither hepatic cytochromes P-450 enzymes nor glutathione transferase is involved in detoxication of tunicamycin or corynetoxin in rats. The beneficial effect of phenobarbitone may be the result of a special kind of phenobarbitone-inducible enzyme which may not be P-450 dependent.

Animals↗

PubMed1982

[Inhibition of the production of infectious particles in cells infected with Junín virus in the presence of tunicamycin].

The antibiotic tunicamycin, an inhibitor of glycoprotein synthesis, suppressed Junin virus multiplication in Vero cells. No infectious virus was formed if tunicamycin was added, at a concentration of 1 microgram/ml, immediately after virus adsorption and remained up to the end of the replicative cycle. Tunicamycin added at 24,48 or 72 hours p.i. immediately stopped virus synthesis. The effect was irreversible. Virus grown in the presence or absence of tunicamycin and labelled with 3H-uridine was purified and layered on a (20-60%) sucrose gradient. The radioactive profile of both gradients was coincident. The total number of cpm associated to the peak gradient of virus grown with tunicamycin represented 70% of the cpm from control virus. Since viral infectivity was 99.4% inhibited in the presence of TM it can be assumed that most of the radioactive particles formed are not infectious. Therefore, in the presence of tunicamycin Junin virus buds normally. From these results we can conclude that carbohydrates are important for viral infectivity but play no role in the transport of viral proteins through cellular membranes.

Animals↗

PubMed1983

Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin.

Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.

Animals↗

PubMed1982

Effect of tunicamycin on insulin binding and on proteoglycan synthesis and distribution in Swarm rat chondrosarcoma cell cultures.

Tunicamycin, an inhibitor of dolichol-diphospho-N-acetylglucosamine formation and hence an inhibitor of N-linked oligosaccharide biosynthesis, suppressed total proteoglycan synthesis by Swarm rat chondrosarcoma chondrocytes without affecting the size of the proteoglycan molecule, its secretion from the cell, or its ability to be retained in the extracellular matrix. In addition, tunicamycin did not substantially alter the ability of the chondrocytes to polymerize glycosaminoglycan onto an exogenous beta-D-xyloside acceptor. A secondary effect of tunicamycin suppression of proteoglycan synthesis was that a lesser amount of newly synthesized proteoglycan diffused from the extracellular matrix into the culture medium. The ability of exogenous hyaluronic acid and proteoglycan to increase the percentage of newly synthesized 35S-proteoglycan in the medium in tunicamycin-treated cultures indicates that matrix retention of 35S-proteoglycan is related to the total extracellular uronic acid content rather than to the presence or absence of mannose oligosaccharides bound to the proteoglycan molecule. These noncytotoxic concentrations of tunicamycin (33-333 ng/ml) decreased [3H]mannose incorporation to the same extent that they decreased total [35S]sulfate and [3H]serine incorporation, and caused the chondrocyte to synthesize and secrete a species of beta-hexosaminidase that was mannose-deficient as assessed by its failure to bind to concanavalin A. The additional finding of decreased insulin binding to tunicamycin-treated chondrosarcoma chondrocytes suggested that the inhibition of proteoglycan synthesis was due to diminution of receptors which respond to stimulatory hormones.

Acetylglucosaminidase↗

PubMed1977

Changes in surface properties of normal and transformed cells caused by tunicamycin, an inhibitor of protein glycosylation.

Normal and virally transformed mouse (3T3) and human (WI-38) cells were treated with tunicamycin, an inhibitor of lipid-carrier-dependent glycosylation of proteins. Incubation of cells with tunicamycin (1 microgram/ml) caused detachment and death of simian virus 40- and polyoma-transformed cells within 24 hr; these effects were not seen with nontransformed cell lines. However, the proliferation of 3T3 cells was inhibited by tunicamycin and, after a few days, a distinct change from an epithelioid to an abnormally elongated shape was observed. Both inhibition of growth and the morphological changes were reversible. A marked decrease in concanavalin A agglutinability was observed in virally transformed cells treated with tunicamycin (0.5 microgram/ml), but agglutination by wheat germ agglutinin or soybean agglutinin was unaffected. Analysis of biosynthetically labeled proteins showed that a high-molecular-weight protein, presumed to be related to fibronectin, is markedly reduced in the medium of cells cultured in the presence of tunicamycin. These results suggest that tunicamycin interferes with the insertion or function of one or more cell-surface glycoproteins. Such cell-surface changes could affect a number of cellular properties, including attachment, cell shape, and agglutinability by some lectins.

Agglutination Tests↗

PubMed1977

Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells.

Tunicamycin, an antibiotic which blocks the formation of N-acetylglucosamine-lipid intermediates, thereby preventing glycosylation of glycoproteins, inhibits the secretion of IgA and IgE by MOPC 315 mouse plasma cells and IR162 rat plasma cells, respectively. At 0.5 microng of tunicamycin per ml, D-[14C]glucosamine incorporation into newly synthesized immunoglobulin was inhibited greater than 90% while the overall rate of protein synthesized was much less inhibited (40% in the case of MOPC 315 cells and 13% in the case of IR162 cells). This dose of tunicamycin produced an 85% inhibition of IgA secretion by the MOPC 315 cells and a complete inhibition of intact IgE secretion by the IR162 plasma cells. In contrast, tunicamycin had little effect on the secretion of normally nonglycosylated lambda light chains or on cell-free protein synthesis, demonstrating that tunicamycin is not a general inhibitor of protein synthesis or a non-specific inhibitor of protein secretion. No enhancement of intracellular degradation of nonglycosylated immunoglobulin could be demonstrated. Electron microscopy of tunicamycin-treated MOPC 315 cells revealed marked dilatations of the rough endoplasmic reticulum, and direct immunofluorescence indicated that the dilated rought endoplasmic reticulum contained IgA. These data indicate that glycosylation of newly synthesized IgA and IgE may be necessary for normal secretion to occur.

Acetylglucosamine↗

PubMed1998

Downregulation and subcellular redistribution of the gamma-aminobutyric acidA receptor induced by tunicamycin in cultured brain neurons.

The significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of gamma-aminobutyric acidA receptor (GABA(A)R) in cultured neurons derived from chick embryo brains. Incubation of cultures with 5 microg/ml of tunicamycin for 24 h blocked the binding of 3H-flunitrazepam and 3H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%, respectively. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABA(A)R immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased to 24% from 34% of control in the presence of the agent, as revealed by trypsinization of cells in situ followed by 3H-flunitrazepam binding. The molecular weight of the receptor subunit protein was lowered around 0.5 kDa after tunicamycin treatment, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABA(A)R by tunicamycin. Moreover, intense inhibitions of 91% and 44%, respectively, were detected to the general galactosylation and mannosylation in the tunicamycin-treated cells, whereas the protein synthesis was hindered by 13%, through assaying the incorporation of 3H-sugars and 3H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 microg/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in the ligand binding. In addition, in situ hybridization analysis showed that these three inhibitors did not perturb the mRNA of GABA(A)Ralpha1-subunit. The data suggest that tunicamycin causes the downregulation and subcellular redistribution of GABA(A)R by producing irregularly glycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation may be important for the functional expression and intracellular transport of GABA(A)R.

Animals↗

PubMed1987

In vivo effects of tunicamycin on the secretory processes of rat parotid glands.

The morphological and functional effects of tunicamycin were studied in rat parotid glands at the stage of the reformation of secretory granules following secretory stimulation by isoproterenol. Tunicamycin inhibited the incorporation of (3H)-mannose into the acid-insoluble fraction but had no effect on total protein synthesis as determined by the incorporation of (14C)-leucine. Thus the administration of tunicamycin in vivo inhibits the synthesis of mannose-rich glycoproteins in a manner similar to that in an in vitro system. The ultrastructure of the acinar cell showed little change following treatment with this drug, except that the number of reaccumulated secretory granules was greater than in the control. Amylase secretion stimulated by isoproterenol was inhibited in tunicamycin-treated cells, but did not decrease following treatment with N6,2'-O-dibutyryladenosine 3'-5'-cyclic monophosphate, a secretory stimulator bypassing the beta-receptor. A radio-receptor assay using (3H)-dihydroalprenolol and direct localization using the fluorescent beta-adrenergic blocker 9-amino-acridin-propranolol showed a marked reduction in the binding activity of beta-receptor following treatment with tunicamycin. Thus the inhibition of N-linked glycosylation appears to produce profound effects on the beta-adrenergic receptor-adenylate cyclase complex of acinar cells, although the steps of the transport and the exocytotic discharge of secretory materials are not affected.

Adenylyl Cyclases↗

PubMed1986

The preparation of tritiated tunicamycin.

A relatively simple and inexpensive procedure was devised for the radiolabeling of the glycoprotein biosynthesis inhibitor, tunicamycin. The procedure is based on hydrogen exchange in alkaline solutions of tritiated water. It was noted that the antibiotic was much more alkali labile than model compounds such as uridine. The alkali stability of the inhibitor was studied to determine conditions for optimum labeling and yield. The effects of alkaline incubation on the inhibitory properties of the antibiotic were also investigated and it was found that the breakdown products are not effective inhibitors of the reaction that transfers N-acetylglucosamine-1-phosphate to dolichyl phosphate. The isolated radioactive tunicamycin homologs, however, retained all their inhibitory action. Incubation of tunicamycin in the presence of deuterated water and mass spectral analysis showed that under the conditions used for the tritiation of tunicamycin the major product exchanged six hydrogen atoms. The position of the tritium atoms in labeled tunicamycin was not determined. The radioactive label in these compounds was shown to be stable under physiological conditions and should be useful for investigations involving the action of these antibiotics.

Alkalies↗

PubMed1985

Effect of tunicamycin on hexose transport in mouse embryo fibroblast Swiss 3T3 cells.

The role of glycosylation of the carrier in the transporting activity was investigated in Swiss 3T3 cells. Inhibition of protein glycosylation by tunicamycin resulted in the decrease of hexose uptake in a dose- and time-dependent manner without a cytotoxic effect. From kinetic analysis, a decrease in the number or availability of hexose carriers in the plasma membrane was suggested. This was in good correlation with the decrease in the amount of photoaffinity cytochalasin B binding in the plasma membrane by the treatment with tunicamycin. The rate of phorbol 12,13-dibutyrate-induced translocation of the hexose carrier from microsomal to plasma membrane was reduced in tunicamycin-treated cells, which may be correlated with the decrease in the number of the completely glycosylated carrier translocatable from the microsomal membrane. In both tunicamycin-treated and untreated cells, the stimulation of hexose transport by phorbol 12,13-dibutyrate was abolished by the removal of phorbol 12,13-dibutyrate, and upon its readdition the stimulation recovered to the same degree as before the removal. Thus, the recycling of the functionally mature hexose carrier appeared not to be affected by the treatment with tunicamycin. These results suggested that complete glycosylation of the carrier may be necessary for the translocation of the carrier from microsomal to plasma membrane to accomplish its function on the cell surface.

3-O-Methylglucose↗

PubMed1988

In vivo effect of tunicamycin on the expression of rat small intestinal brush border membrane glycoproteins and glycoenzymes.

Tunicamycin, a known inhibitor of the lipid-dependent glycosylation of proteins, was used in vivo to study the biosynthesis of rat intestinal brush border membrane aminopeptidase N and dipeptidyl aminopeptidase IV. The incorporation of [3H]glucosamine into newly synthesized total protein of mucosal cell homogenates was inhibited by 60%, whereas incorporation of [3H]leucine was decreased only 21% by tunicamycin. This effect was much more pronounced in the brush border membrane fraction isolated from intestinal mucosal cells where incorporation of radiolabled leucine and glucosamine was reduced to 50 and 82% of control values respectively. An examination of the brush border membrane protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there was a marked selective decrease in the amount of glycoproteins of molecular weights greater than 130 kD. In addition, there were decreased levels of assayable aminopeptidase N, dipeptidyl aminopeptidase IV and disaccharidase activity in intestinal mucosal cell homogenates and brush border membranes of tunicamycin-treated rats. Though tunicamycin decreased incorporation of newly synthesized aminopeptidase N and dipeptidyl aminopeptidase IV protein into brush border membranes by 70-75%, the newly synthesized enzyme that was incorporated was indistinguishable from that of controls. Further, non-glycoslyated forms of both enzymes were not detected in any other subcellular fractions. These results show that tunicamycin, an inhibitor of glycosylation, significantly affected the expression of brush border membrane glycoproteins, suggesting that both polypeptide synthesis and degradation of these proteins may be altered in the presence of this drug.

Aminopeptidases↗

PubMed1989

Effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of acidic amino acid neurotransmitters in C6 glioma cells.

The effect of tunicamycin, an inhibitor of protein glycosylation, on the high-affinity transport of D-aspartate was investigated in C6 astrocytoma cells. A concentration of tunicamycin (1 microgram/ml) that after 24 h exposure inhibited the rate of transport by 70% and incorporation of [3H]mannose by 82-95% had only a small effect on [14C]leucine incorporation into protein and cell growth (20% reduction). Tunicamycin decreased the Vmax for transport without affecting the Km, which suggests that inhibition of glycosylation reduces the number of competent transporters on the surface of the plasma membrane. The decrease in the velocity of uptake was attenuated when C6 cells were treated with tunicamycin in the presence of protease inhibitors, indicating that the underglycosylated carriers are subject to enhanced proteolytic degradation. Incubation in drug-free medium following treatment with 1 microgram/ml of tunicamycin for 24 h resulted in recovery of D-aspartate transport within 48 h. This recovery was prevented by the presence of cycloheximide, which indicates that synthesis of new transporters is necessary for the restoration of normal rates of D-aspartate uptake. These results support our earlier postulate that the high-affinity carriers for amino acid transmitters are transmembrane glycoproteins.

Amino Acids↗

PubMed1983

Role of glycoproteins in neuronal differentiation. Inhibition of neurite outgrowth and the major cell surface glycoprotein of murine neuroblastoma cells by a purified tunicamycin homologue.

Mouse neuroblastoma cells in culture can be induced to differentiate morphologically by serum deprivation or by dibutyryl cyclic AMP (db-cAMP), e.g. they appear flattened, adhere more firmly to the culture substratum and extend long neuritic processes, and thus represent a widely used model system for neuronal cells. This differentiation is accompanied by modulation of cell surface components, such as the induction of a high molecular weight (HMW) glycoprotein (200 kD). We have studied the role of glycoproteins in the process of neuronal differentiation, using a purified homologue of the antibiotic tunicamycin (Al-tunicamycin) and neuroblastoma N115 cells grown in culture. Al-tunicamycin markedly inhibited (up to 60-75%) the incorporation of radioactively labelled sugars into cellular proteins of differentiating neuroblastoma cells. Concomitantly, the cells altered their morphology, they became rounded and less adhesive and retracted their neurites. Changes in the appearance, glycosylation and electrophoretic mobility of several cellular and secreted glycoproteins were observed, when cells were incubated in the presence of Al-tunicamycin. The most striking effect of Al-tunicamycin on the composition of cellular glycoproteins was the marked reduction in appearance of the 200 kD glycoprotein. The findings suggest that glycoproteins and in particular the neuron-specific 200 kD glycoprotein, are related to morphological differentiation processes, mainly to cellular adhesion and neurite outgrowth.

Animals↗

PubMed1979

Glycoprotein synthesis and inhibition of glycosylation by tunicamycin in preimplantation mouse embryos: compaction and trophoblast adhesion.

The synthesis of glycoproteins and inhibition of protein glycosylation by tunicamycin were examined during development of preimplantation mouse embryos and trophoblast adhesion. Tunicamycin specifically inhibits glycosylation of asparaginyl residues of glycoproteins. Tunicamycin, 0.25-5.0 microgram/ml, had no effect on early cleavage or aggregation between embryos, but the embryos remained irreversibly uncompacted when control embryos developed to the blastocyst stage. Trophoblast adhesion and giant cell outgrowth were reversibly inhibited and the binding of Con A was also reduced. Incorporation of 3H-mannose into blastocysts was inhibited by 80%, but that of 3H-glucosamine and 3H-leucine by only 28 and 18%, respectively, in the presence of 1.0 microgram/ml tunicamycin. Qualitative analysis showed that the incorporation of the sugars was markedly reduced in the majority of the fractions, but the synthesis of these carbohydrate-deficient glycopeptides was essentially normal. However, protein-polysaccharide fractions with nearly 40% of the incorporated glucosamine and only 5% mannose and 1% leucine were insensitive to inhibition by tunicamycin. Membrane-bound N-glycosidically linked glycoproteins therefore evidently play an important role during compaction and in trophoblast adhesion of mouse embryos.

Animals↗

PubMedDate not supplied

The effects of tunicamycin on secretion, adhesion and activities of the cellulase complex of Clostridium cellulolyticum, ATCC 35319.

The effects of tunicamycin, an inhibitor of N-asparagine-linked glycosylation, on the secretion, adhesion and activities of the cellulase complex produced by Clostridium cellulolyticum have been studied. Tunicamycin at 0.1 micrograms/ml slightly inhibited growth on cellobiose. Endoglucanase, p-nitrophenylcellobiosidase and avicelase activities of the "Avicel"-adsorbed fraction from a culture grown with this drug were decreased 4.4-, 1.4- and 12.2-fold, respectively. During growth on cellulose, tunicamycin considerably inhibited growth and adhesion of cells on their substrate (only 28% of the cells were bound to cellulose). SDS-PAGE mobilities of some proteins excreted during growth with the drug were different from those of proteins from control cultures; the native Avicel-adsorbed fraction (PH2O) consisted of three major components of molecular weights about 135, 90 and 68 kDa, whereas in the presence of tunicamycin (0.1 micrograms/ml), the Avicel-adsorbed fraction (PH2OT) contained only a major band of 105 kDa, and the proteins of 135 and 68 kDa appeared weakly. By using the "Dig Glycan Detection" kit, some proteins appeared to be glycosylated, such as the 135-, 95-, 47- and 40-kDa proteins. Moreover, the affinity for Avicel and the avicelase activity decreased dramatically for the Avicel-adsorbed fraction from a culture grown with the drug. The remaining avicelase activity of the PH2O fraction in the presence of specific P135 antiserum was 50% of the initial activity, whereas CMCase and pNPCbase were not affected. The glycosylated protein of 135 kDa played a prominent role in the adhesion and avicelase activity of C. cellulolyticum. Moreover, the endoglucanase activity in a culture broth from tunicamycin-grown cells was more thermolabile and protease-sensitive than that from control cultures.

Anti-Bacterial Agents↗

PubMedDate not supplied

Photoreceptor-specific degeneration caused by tunicamycin.

The antibiotic tunicamycin inhibits the biosynthesis of N-acetylglucosaminylpyrophosphoryl polyisoprenol, a key intermediate in the formation of the asparagine-linked oligosaccharides of glycoproteins. The effects of tunicamycin have been studied in various biological systems, primarily with the aim of elucidating the role of the carbohydrate moieties in the cellular function of glycoproteins. Rhodopsin, the visual pigment of retinal rod photoreceptor cells, is a membrane glycoprotein which consists of a single polypeptide chain (opsin) to which a chromophoric prosthetic group (II-cis-retinaldehyde) and two asparagine-linked oligosaccharide chains are covalently attached. The glycosylation of opsin can be blocked with tunicamycin in vitro in conditions where polypeptide synthesis is only slightly decreased. We have reported that tunicamycin can disrupt the normal assembly of rod outer segment membranes in vitro without significantly inhibiting the biosynthesis or intracellular transport of opsin. Here we report that intraocular injection of tunicamycin produces a photoreceptor-specific degeneration characterized by progressive shortening of rod outer segment, decreased membrane assembly, and eventual photoreceptor cell death.

Animals↗

PubMed1979

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo fibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (alpha-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.

Amino Acids↗