Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in cultured fibroblasts

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/μg DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/μg DNA). After incubation of fibroblasts with [³H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [³⁵S]sulfate resulted in the incorporation of [³⁵S]sulfate into 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [³⁵S]sulfate and [³H]glucosamine from 5 min to 16 h showed that [³⁵S]PAPS was equilibrated in less than 10 min, while [³H]glucosamine required a longer time, 2–4 h, to attain a steady state with UDP-N-acetylhexosamine. [¹⁴C]Glucose required approximately the same time as [³H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.     

The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

d-[1-¹⁴C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1-¹⁴C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1-¹⁴C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Glucosamine metabolism in regenerating rat liver.

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.     

Effects of Several Tunicamycin-like Antibiotics on Glycoprotein Biosynthesis in Mung Beans and Suspension-cultured Soybean Cells

The antibiotics Streptovirudin and 24010 were tested to determine their effects on the formation of lipid-linked saccharide intermediates associated with glycoprotein biosynthesis in mung bean (Vigna radiata) and suspension-cultured soybean cells (Glycine max cv. Mandarin). In vitro both compounds strongly inhibited the transfer of N-acetyl[³H]glucosamine from UDP-N-[³H]acetylglucosamine to N-acetylglucosaminyl-pyrophosphoryl-polyisoprenol and lipid-linked oligosaccharides, although they had no apparent effect on the incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol with a small inhibition into lipid-linked oligosaccharides. In vivo, Streptovirudin and tunicamycin dramatically inhibited the incorporation of N-[¹⁴C]acetylglucosamine and [³H]mannose into Pronase-released material (glycoproteins), whereas there was no effect on [³H]leucine incorporation into Pronase-released material (protein). Because the action of Streptovirudin and antibiotic 24010 in plants and other systems is similar to that for tunicamycin, these antibiotics are believed to be closely related. The use of tunicamycin is discussed with respect to its importance in studying glycoprotein biosynthesis and function in animal and plant systems.     

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1. When pig ear skin slices were cultured for 18h in the presence of 1μg of tunicamycin/ml the incorporation of d-[³H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5μg of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-¹⁴C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[³H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[³H]glucosamine but not of (U-¹⁴C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[³H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[³H]glucosamine and ³⁵SO₄²⁻ into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

The tissue content and turnover rates of intermediates in the biosynthesis of glycosaminoglycans in young rat skin

1. The tissue contents of hexose monophosphate, N-acetylglucosamine 6-phosphate, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-glucuronic acid were determined in the skin of young rats less than 1 day post partum. Tissue-space determinations were used to calculate their average cellular concentrations. 2. The incorporation of [U-¹⁴C]-glucose into the intermediates was recorded with time and their rates of turnover were calculated. The results demonstrated product–precursor relationships along the pathway of hexosamine synthesis and that of hexuronic acid synthesis. The rates of synthesis of UDP-N-acetylhexosamine and UDP-glucuronic acid were 1·5±0·3 and 0·24±0·03mμmoles/min./g. of tissue respectively. These results indicated the average turnover time of the total tissue glycosaminoglycans to be about 5 days.     

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Procedures for the preparation of UDP-N-[1-¹⁴C]acetyl-d-glucosamine and UDP-N-[1-¹⁴C]acetyl-d-galactosamine with very high specific activities are deseribed. The overall yield based on the amount of [1-¹⁴C]acetate used is greater than 80%. The N-acetyl-d-glucosamine-α-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-d-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-d-glucosamine-α-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. d-glucosamine-α-1-phosphate is N-acetylated with [¹⁴C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-d-glucosamine pyrophosphorylase. UDP-N-[1-¹⁴C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-¹⁴C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

A procedure for the synthesis of uridine-5'-diphospho-N-acetylglucosamine-14C

A complete procedure for the synthesis of 1-¹⁴C-glucosamine-labeled UDP-N-acetylglucosamine is described. Glucosamine is first phosphorylated with ATP and hexokinase to form glucosamine 6-phosphate. This is N-acetylated with acetic anhydride, and the product is converted to UDP-N-acetylglucosamine by incubation with a crude yeast extract. The sugar nucleotide is isolated from the incubation mixture by paper electrophoresis, and purified by paper chromatography.     

Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Effects of ADP, ethanol and acetaldehyde on the relaxing complex of human muscle and its adsorption by polystyrene particles.

Protoplasts of Saccharomyces strain 1016 took up [³H]glucosamine in the presence of an energy source; mannose was chosen to minimize randomization. It accumulated in the soluble intracellular pool primarily as UDP-N-acetyl[³H]glucosamine along with a small amount of [³H]glucosamine 6-phosphate. The antibiotic tunicamycin (TM) at 10 μg/ml did not affect the levels of these metabolites or inhibit the formation of the Nacetylglucosamine polymer, chitin, but did prevent the incorporation of [³H]glucosamine into mannan peptides and the synthesis of invertase. In vitro incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannan in a membrane preparation was not sensitive to 100 μg of TM/ml. TM appears to inhibit an N-acetylglucosaminyl transferase essential for glycoprotein biosynthesis. Binding of [³H]TM reflects its association with the plasma membrane fraction. This material could be recovered in an unaltered form by extraction with chloroform/methanol. If 0.2% phosphatidyl choline or phosphatidyl serine was added simultaneously with the [³H]TM, the binding of [³H]TM was greatly reduced, and the inhibitory effects of TM on protoplasts were prevented; however, addition of phospholipid 20 min later did not eliminate the inhibition, although about 80% of the bound [³H]TM was removed. TM interacts with lipophilic membrane components as well as inhibiting glycoprotein synthesis.     

Studies on N-glycosylation by elongating tissues and membranes from pea stems

Glucosamine and mannose were incorporated into oligosaccharides linked to either polar membrane-lipids or to asparagine residues of endogenous proteins in apical growing tissues of the etiolated pea stem. The glycolipids were subject to turnover in pulse-chase tests and protein-linked oligosaccharides accumulated with time, as expected for a precursor-product relationship. The newly formed glycoproteins were hydrolyzed by endo-β-N-acetylglucosaminidase H to oligosaccharides in the same size range as those released by dilute acid from the lipid-linked oligosaccharides formed during the pulse. The glycoproteins were also partly degraded to free N-acetylglucosamine by β-N-acetylhexosaminidase. Affinity of the carbohydrate moiety of the protein for concanavalin A increased between the beginning and the end of the chase, indicating processing following core glycosylation.

The addition of UDP-N-acetyl-[¹⁴C]glucosamine plus external peptide acceptors (derived from carboxymethylated α-lactalbumin) to membrane preparations from the pea stem resulted in peptide glycosylation at the expense of lipid-linked oligosaccharide. Glycosylation of endogenous protein acceptors did not take place via lipid intermediates but directly from the sugar nucleotide substrate. Tunicamycin inhibited glycosyltransfer to both glycolipids and added peptides, but not to endogenous protein. It is concluded that limiting factors for N-glycosylation by pea membranes in vitro could include the unavailability of endogenous acceptors or the inability to fully elongate and internalize lipid precursors, but is not due to any limitation in capacity for N-glycosylation.

     

Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L

Developing pea cotyledons incorporate radioactivity in vivo from [¹⁴C]glucosamine and [¹⁴C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [¹⁴C]glucosamine and [¹⁴C]mannose is also incorporated into glycopeptides. Considerable redistribution of [¹⁴C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N-acetylglucosamine to asparagine glycopeptide linkage has been isolated from [¹⁴C]glucosamine-labeled glycoproteins.     

Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH¹⁴CO₃ in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH¹⁴CO₃ and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the relative degradation rates of individual species which suggest that the synthesis and degradation of the various surface membrane glycoproteins proceed at rates that are controlled independently.