EVIDENCE FOR THE BIOSYNTHESIS OF MANNOSYLPHOSPHORYLDOLICHOL AND N-ACETYLGLUCOSAMINYLPYROPHOSPHORYLDOLICHOL BY AN AXOLEMMA-ENRICHED MEMBRANE PREPARATION FROM BOVINE WHITE MATTER

An axolemma-enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [¹⁴C]mannose and N-acetyl[¹⁴C]glucosamine from their nucleotide derivatives into a mannolipid and an N-acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N-acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma-enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP-[¹⁴C]mannose or UDP-N-acetyl[¹⁴C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma-enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma-enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N-acetylglucosaminyl lipid synthesis were compared for axolemma-enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma-enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three-fold lower. The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP-mannose and UDP-N-acetylglucosamine has also been demonstrated in the axolemma-enriched membrane fraction.     

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.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

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.     

Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Biosynthesis of glycosoaminoglycans by microsomal preparations from cultured mastocytoma cells

Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of ¹⁴C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3′-phosphate 5′-sulphatophosphate, as well as UDP-[¹⁴C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.     

Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [¹⁴C] glucosamine catalyzes the tranfer of N-acetylglucosamine to an endeenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimulated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine.The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose.The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[¹⁴C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.     

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.     

Glycosylation of endogenous lipids and proteins by preparations of chicken embryo fibroblasts

Glycosylation of endogenous phosphoisoprenyl lipids and membrane-associated proteins was shown to occur in preparations of chicken embryo fibroblasts incubated with GDP[¹⁴C]mannose and UDP-N-acetylglucosamine. The two preparations used were cells released from the culture dishes by buffered saline containing EDTA and crude membranes from those cells. Both β-mannosyl-phosphoryldolichol and oligosaccharide-phosphoryl lipids with five to eight sugar residues were labelled under the conditions employed. The oligosaccharide isolated from the octasaccharide-lipid fraction was shown to be heterogeneous after an analysis of the products formed by treatment of the oligosaccharide with glycosidases. Some of the oligosaccharides appeared to contain N-acetylglucosamine at positions external to that of [¹⁴C]mannose. Lipids with oligosaccharide moieties of different structures were made by the two preparations. The results of pulse-chase experiments were consistent with the glycosylated lipids being intermediates in glycoprotein biosynthesis.     

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.     

The enzymic synthesis of β-[32P]UDP-N-acetylglucosamine

Cells of Micrococcus sp. 2102 incorporate inorganic [³²P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[³²P]phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP: N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise β-[³²P]UDP-N-acetylglucosamine.     

Glycoprotein biosynthesis in plants. Formation of lipid-linked oligosaccharides of mannose and N-acetylglucosamine by mung bean seedlings.

Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNAc from UDP-[³H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[¹⁴C]mannose and UDP-[³H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates.     

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.     

Synthesis and characterization of lipid-linked mannosyl oligosaccharides in Volvox carteri f. nagariensis

Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[¹⁴C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphospnate-chitobiose-(mannose)₅ structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 314 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [¹⁴C]glycolipids, revealed that the first mannose is bound via a β-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as α-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost α-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.     

Synthesis in vitro of glycoprotein in regenerating rat liver

The metabolism of glucosamine in regenerating rat liver was studied in liver slices. [1-¹⁴C]Glucosamine was incorporated into acid-soluble fraction, rapidly converted to UDP-N-acetylhexosamine and transferred to acid-insoluble fraction. Electrophoretic analysis revealed that most of the radioactive macromolecules released from the slices to the incubation medium were plasma glycoproteins.The incorporation of [1-¹⁴c]glucosamine into UDP-N-acetylhexosamine significantly increased from 6 h to 48 h after partial hepatectomy. On the contrary, the incorporation into acid-insoluble fractions of slice and medium decreased to about 50% of the control values. The rate of transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to acid-insoluble fractions also decreased at 12 h and 48 h respectively. This indicates that the transfer of N-acetylhexosamine to glycoproteins decreases during 48 h of liver regeneration.The enhancement of [1-¹⁴C]glucosamine incorporation into UDP-N-acetylhexosamine is due to an accumulation of the label in the larger pool of this compound. Evidently, some control mechanism may operate on the transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to glycoproteins in regenerating rat liver.     

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.     

Dolichylphosphate-dependent Glycosyl Transfer Reactions in the Endoplasmic Reticulum of Castor Bean Endosperm

In the endosperm of Ricinus communis (castor bean) a number of glycosyl transferases were found to be present during germination. They catalyze the incorporation of mannose from guanosine diphosphate mannose and of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine into a glycolipid fraction, which had all of the properties of dolichylphosphate and pyrophosphate sugars, respectively. The sugar moiety of dolichylphosphate mannose is transferred to a lipid-oligosaccharide, containing more than 6 hexose units. When the membranes are preincubated with nonradioactive guanosine diphosphate mannose and uridine diphosphate N-acetylglucosamine, radioactivity from dolichylphosphate [¹⁴C]mannose is also transferred to a glycopolymer. In addition, the formation of radioactive glycoproteins from guanosine diphosphate [¹⁴C]mannose has been demonstrated using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography.     

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.     

Glycoprotein synthesis in plants: I. Role of lipid intermediates

The enzymic processes involved in glycoprotein synthesis have been studied using crude extracts obtained from developing cotyledons of Phaseolus vulgaris harvested at the time of active deposition of vicilin. Radioactivity from GDP-[¹⁴C]mannose can be incorporated by crude extracts into a single chloroform-methanol-soluble product as well as into insoluble product(s). Mannose is the sole ¹⁴C-labeled constituent of the lipid. The kinetics of incorporation of ¹⁴C, as determined by pulse and pulse-chase experiments using GDP-[¹⁴C]mannose, as well as direct incorporation from added [¹⁴C]mannolipid, shows that the mannolipid is an intermediate in the synthesis of the insoluble product(s). The characteristics of the mannolipid are consistent with it being a mannosyl phosphoryl polyprenol. The mannose is apparently attached to the lipid via a monophosphate linkage. Of the radioactivity in the insoluble product(s), about 20% is pronase-digestible during a “pulse experiment.” After a chase with unlabeled GDP-mannose, about 40% is pronase-digestible; the other 60% is as yet uncharacterized. A radioactive product soluble in a mixture of chloroform-methanol-H₂O can be extracted from the insoluble residue obtained during a pulse, but is no longer present after a chase. This product may be a lipid oligosaccharide, the final intermediate in glycoprotein synthesis. Data are presented on incorporation from UDP-N-[¹⁴C]acetylglucosamine into both chloroform-methanol-soluble and -insoluble product(s). The results are consistent with an involvement of lipid intermediates in the glycosylation of protein in this system, and support the concept that the mechanisms of glycoprotein synthesis in higher plants are similar to those which have been reported for mammalian systems.