Search

The binding constants of N-acetylglucosamine (G1cNAc) and its methyl alpha- and beta- glycosides to hen and turkey egg-white lysozymes [EC 3.2.1.17], in the latter of which Asp 101 is replaced by Gly, were determined at various pH values by measuring changes in the circular dichroic (DC) band at 295 nm. The binding of beta-methyl-G1cNAc to turkey and hen lysozymes perturbed the pK value of Glu 35 from 6.0 to 6.5, the pK value of Asp 52 from 3.5 to 3.9, and the pK value of Asp 66 from 1.3 to 0.7. In addition, perturbation of the pK value of Asp 101 from 4.4 to 4.0 was observed in the binding of this saccharide to hen lysozyme. The binding of alpha-methyl-GlcNAc to hen and turkey lysozymes perturbed the pK value of Glu 35 to the alkaline side by about 0.5 pH unit, the pK value of Asp 66 to the acidic side by about 0.5 pH unit, and the pK value (4.4) of an ionizable group to the acidic side by about 0.6 pH unit. The last ionizable group was tentatively assigned to Asp 48. The pK value of Asp 52 was not perturbed by the binding of this saccharide. The pH dependence curves for the binding of GlcNAc to hen and turkey lysozymes were very similar and it was suggested that Asp 48, in addition to Asp 66, Asp 52, and Glu 35, is perturbed by the binding of GlcNAc.

Laser Raman spectra of α- d -glucose, D-glucosamine · HCl, N- acetyl- d -glucosamine and d -glucuronic acid were obtained. The vibrations originating from OH and NH functional groups were identified on the basis of the deuterium exchange results. By comparing the spectra of α- d -glucose, d -glucuronic acid, d -glucosamine · HCl, and N- acetyl- d -α- glucosamine , the vibrations due to COOH, CH 2 , NH 3 + , and =NH groups were also identified.

The distribution of radioactivity in L7 ventral roots after microinjection of N-acetyl-3H-glucosamine into the L7 segment of the feline neuraxis was examined under several conditions; i.e., the isotope was injected before and after ligation of the roots and various periods were allowed for uptake. The results indicated a transport of radiolabeled macromolecular material in both slow and fast components of axoplasmic flow. Further characterization of the nature of the transported, macromolecular material revealed that more than half of the radioactivity was non-ultrafilterable even after pronase digestion, only 2% of this activity was incorporated into acidic glycosaminoglycans and the remaining activity was probably derived from glycoproteins.

The effect of glucosamine and N-acetylglucosamine on aggregation and energy metabolism was investigated over an 8-h period in cells dissociated by 0·25 % (w/v) trypsin from the skeletal muscle of 9-day-old chick embryos. At 8 h, 0·023 M glucosamine and N-acetylglucosamine inhibited the aggregation of cells suspended in Eagle’s minimal essential medium by 18·9 % and 16·4% respectively, as judged on a basis of aggregate size. Glucosamine and N-acetylglucosamine reduced the cellular ATP level by a mean of 36 % and 27 % respectively; values reflected in the 32% and 19% loss of total adenine nucleotides caused by these sugars. The adenine nucleotide balance was also changed from a mean control ATP/AMP ratio of I3·7 to 8·75 by N-acetylglucosamine and to 8·0 by glucosamine. Intracellular lactate/pyruvate ratios were similarly disrupted in cells incubated with 0·023 M hexosamine. Although the hourly values fluctuated, it was seen that the amount of lactic acid relative to pyruvic acid, considered as an average for the 8-h period, was raised from 6:1 in controls to 8:1 in N-acetylglucosamine-treated and to 10:1 in glucosamine-treated cell preparations. Compared to controls at 8 h, glucosamine enhanced the production of lactate into the suspension medium by 99%. The N-acetyl analogue caused cells to produce more lactic acid than did controls for 4–5 h only, for by 8 h 25 % less of this metabolite was assayed in the culture medium. The incorporation of D-[U-14C]glucose into glycogen paralleled the results of extracellular lactic acid assays. N-acetylglucosamine inhibited the incorporation by 30 % at 4 h, although by 6 h, and for the remainder of the experimental period, there was more 14C-labelled glycogen in these cells than in controls. By contrast, glucosamine inhibited the incorporation of radioactive glucose into glycogen by 42% at 4 h and, unlike N-acetylglucosamine, consistently thereafter. Glucosamine also enhanced cellular oxygen uptake throughout the experimental period, to the extent of 59 % at 8 h. The oxygen uptake of N-acetylglucosamine-treated cells was similar to controls until about the 5th hour, when there was a subsequent inhibition which had accumulated to 13 % by the end of the experiment. The release of 14COs by cells was inhibited by glucosamine. This hexosamine depressed production by 19% at 12 h whereas N-acetylglucosamine inhibited this evolution by 9 % at this time. The metabolic effects of these hexosamines on chick muscle cells in vitro are mainly attributed to a central alteration of the adenine nucleotide balance although certain other documented effects of glucosamine are considered to be involved. An inhibition of cell aggregation by glucosamine and N-acetylglucosamine is discussed in terms of a depressed cellular metabolic economy.

1. The synthesis of a polymer of N-acetylglucosamine 1-phosphate, occurring in the walls of Staphylococcus lactis N.C.T.C. 2102, was examined by using cell-free enzyme preparations. The enzyme system was particulate, and probably represents fragmented cytoplasmic membrane. 2. Uridine diphosphate N-acetylglucosamine was the only substrate required for polymer synthesis and labelled substrate was used to show that N-acetylglucosamine 1-phosphate is transferred as an intact unit from substrate to polymer. 3. The properties of the enzyme system were studied. A high concentration of Mg2+ or Mn2+ was required for optimum activity, and the pH optimum was about 8·5. 4. End-group analysis during synthesis in vitro showed that newly formed chains contain up to about 15 repeating units. Pulse-labelling indicated that chain extension occurs by transfer from the nucleotide to the ‘sugar-end’ of the chain, i.e. to the end that is not attached to peptidoglycan in the wall.

An investigation of the structure and peptide attachment of the mannose-N-acetylglucosamine unit (unit A) from human and calf thyroglobulin has been carried out on glycopeptides containing large (8 to 10 mannose plus 2 glucosamine residues) and small (5 to 6 mannose plus 2 glucosamine residues) variants of this unit, as well as on an oligosaccharide fraction (8 mannose plus 2 glucosamine residues). All of the mannose residues could be released by α-mannosidase, after which 1 N-acetylglucosamine could be removed by β-N-acetyglucosaminidase. This permitted the isolation from the glycopeptides of 2-acetamido-4-O-(2acetamido-2-deoxy-β-d-glucopyranosyl)-1-N-β-l-aspartyl-2deoxy-β-d-glucopyranosylamine and 2-acetamido-1-N-β-laspartyl-2-deoxy-β-d-glucopyranosylamine, which could be split to aspartic acid plus di-N-acetylchitobiose or N-acetylglucosamine, respectively, by the action of glycosyl asparaginase. Incubation of the unit A oligosaccharide with α-mannosidase resulted in the production of a di-N-acetylglucosamine disaccharide in high yield which was shown to be identical with di-N-acetylchitobiose. Serial periodate oxidation of the glycopeptides caused destruction of all but approximately 2 of the mannose residues, but spared both N-acetylglucosamine residues in the first Smith degradation. These 2 surviving mannose residues were destroyed in the second degradation. One glucosamine was oxidized in the third treatment, while the other glucosamine still remained intact. Glycerol was the only alcohol released during the three Smith degradations. Periodate oxidation and sodium borohydride reduction of the reduced unit A oligosaccharide, in which the terminal glucosamine had been converted to glucosaminitol, yielded 1 residue of xylosaminitol. Methylation of the glycopeptides by the method of Hakamori yielded tetra- and di-O-methyl mannose ethers from glycopeptides with small carbohydrate units, while tetra-, tri-, and di-O-methyl ethers were obtained from the glycopeptides with large carbohydrate units. All glycopeptides yielded only 1 O-methyl ether of N-methylglucosamine, which was identified by ion exchange chromatography to be 3,6-di-O-methyl-N-methylglucosamine. The results of these studies suggest the following structure for carbohydrate unit A. [see PDF for sequence] The smallest unit found was a tightly branched structure containing 5 mannose residues, and the larger units are believed to contain additional mannose residues, designated as (Man)x linked to the terminal residues of the three chains by α1-2 bonds. No significant species variations were observed between the calf and human proteins.

In the first part of this Discussion the beautiful crystallographic results obtained at the Royal Institution were described (Blake, Mair et al ., p. 365, Blake, Johnson et al ., p. 378). Particular attention was paid to the nature of certain lysozymesaccharide complexes, some involving oligosaccharides of N -acetylglucosamine. This paper describes reactions of these same sugars, namely, their binding and cleavage by the enzyme.

The reaction catalyzed by galactosyltransferase has been studied kinetically at pH 8.0 using glucose as the galactosyl group acceptor in the presence of a fixed concentration of α-lactalbumin. The initial velocity patterns and the patterns for the dead-end inhibition by UDP-glucose were similar to those obtained with N-acetylglucosamine as a substrate in the absence of α-lactalbumin (in an accompanying paper). l -Arabinose acted as a dead-end inhibitor giving rise to inhibitions which were competitive with respect to glucose and uncompetitive in relation to both Mn2+ and UDP-galactose. These results have been interpreted to indicate that there is an ordered addition of Mn2+, UDP-galactose, and glucose to the enzyme, as well as an ordered release of lactose and UDP. It is postulated that Mn2+ does not dissociate from the enzyme-manganese complex during the catalytic reaction. Product inhibition by lactose was not observed. Comparisons have been made of the values for the kinetic constants associated with Mn2+ and UDP-galactose in the absence and presence of α-lactalbumin.

The β(1→4)-linked tetrasaccharide derived from N -acetylglucosamine binds to lysozyme differently than the trimer. The apparent enthalpy of binding of the tetramer is 2.8 kcal/mole less negative than that of the trimer. Because of enthalpy-entropy compensation, the free energy of binding of tetramer and trimer differ only slightly (by 0.3 kcal at 25°C). Consideration of these results in conjunction with other properties of lysozyme suggests that there are two tetramer-lysozyme complexes, one of which has the characteristics of the trimer-lysozyme complex. The pentamer and hexamer of N -acetylglucosamine bind like tetramer.

1. 1. Sindbis-virus-infected chick embryo fibroblasts show an increase in transferase activity of N-acetyl-glucosamine on endogenous protein acceptors. 2. 2. Hypertransglycolysation is a basic cellular reaction to viral infection. 3. 3. Membranous structures are directly involved in this phenomenon.

The influence of glucosamine, galactosamine, glucuronic acid and of some derivatives of these compounds on mucopolysaccharide metabolism and on protein metabolism of primary fibroblast monolayer cultu

The title compound, used in the synthesis of glycopeptides and as a reference substance in the structural elucidation of glycoproteins, was synthesized by condensation of 2,3,4,6-tetra- O -acetyl-α- d -mannopyranosyl bromide with 2-acetamido-4,6- O -benzylidene-α- d -glucopyranosyl azide, followed by removal of the benzylidene group to give the disaccharide azide 6 and acetylation. The resulting fully acetylated disaccharide azide 7 was also obtained by treatment of the known 2-acetamido-1,4,6-tri- O -acetyl-2-deoxy-3- O -(2,3,4,6-tetra- O -acetyl-α- d -mannopyranosyl)-α- d -glucopyranose with hydrogen chloride and then with silver azide. The azide 7 was reduced in presence of platinum oxide (Adams' catalyst), and the resulting amine was condensed with 1-benzyl N -benzyloxycarbonyl- l -aspartate in the presence of N , N ′-dicyclocarbodiimide. The removal of the protective group was accomplished by hydrogenolysis and O -deacetylation. In a second route, the disaccharide azide 6 was reduced and then condensed with 1-benzyl N -benzyloxycarbonyl- l -aspartate, and the resulting product hydrogenolyzed.

Walton, Robert B. (Merck Sharp & Dohme Research Laboratories, Rahway, N.J.) and Edward L. Rickes . Reversal of the antibiotic, bacillin, by N -acetylglucosamine. J. Bacteriol. 84: 1148–1151. 1962.—Antibacillin, previously shown to reverse the antibiotic action of bacillin, was isolated from powdered milk and identified as N -acetylglucosamine, a substance that is present in almost all microbial and animal cells.

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.

1. The activity of the enzyme that splits 2-acetamido-1-l-β-aspartamido-1,2-dideoxy-β-d- glucose (1-aspartamido-β-N-acetylglucosamine) was measured in tissues from different mammalian species. 2. The enzyme from an aqueous extract of rat liver was purified 150-fold in 56% yield. 3. Optimum activity for the hydrolysis of 1-aspartamido-β-N-acetylglucosamine was at pH7, and ammonia and N-acetylglucosamine were liberated in equimolar amounts. At pH8·5, 1-amino-N-acetylglucosamine was the only sugar produced after short periods of incubation. On prolonged incubation there was spontaneous liberation of ammonia from this compound. 4. It is concluded that the enzyme is an amidase.

The association constants with lysozyme of several saccharides containing N-acetyl-d-glucosamine and N-acetylmuramic acid, have been determined by fluorescence spectroscopy or, in one case, by equilibrium dialysis experiments. The association data may be understood on the assumption that the active site of lysozyme consists of a number of subsites binding individual 2-acetamido-2-deoxyglucopyranose rings. On the basis of these data, we have drawn detailed conclusions about the specificity of the individual subsites and their contributions to the total binding energy of oligosaccharide substrates and inhibitors. These conclusions are consistent with, and offer further support of, the mechanism of lysozyme action deduced by Phillips and his coworkers on the basis of x-ray crystallographic studies.

Sepharose-2-acetamido-N-(ϵ-aminocaproyl)-2-deoxy-β-D-glucopyranosylamine was prepared by a reaction of 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-β-D-glucopyranosylamine and N-(benzyloxycarboxyl)-ϵ-aminocaproic acid, removal of the 0-acetyl and the benzyloxycarboxyl groups and coupling to Sepharose. The product was used for the purification of wheat germ agglutinin, by adsorption from a crude wheat germ extract and elution with 0.1M acetic acid. The purified agglutinin was homogeneous on SDS-polyacrylamide gel electrophoresis and had a specific hemagglutinating activity of 3000 u/mg when tested on trypsinized rabbit erythrocytes. It was rich in cysteine, cystine and glycine, and contained no sugar.

Blood group A active ceramide hexasaccharide devoid of N -acetylglucosamme was isolated from lipid extract of hog stomach mucosa. By partial acid hydrolysis, sequential hydrolysis with glycosidases and methylation analysis the structure of this glycolipid was identified as GalNAc−(l → 3)−Gal−(l → 3)−Gal−(l → 4)−Gal−(l → 4)−Glc⊂l → l)−Cer (2↑1)α Fuc

1. Walls of Staphylococcus lactis 2102 contain about 40% of a phosphorylated polysaccharide, which was isolated by extraction with cold trichloroacetic acid, with dilute NaOH, and also by digestion with a Flavobacterium peptidase. 2. The purified polymer contained equimolar proportions of N-acetylglucosamine and phosphate as its sole constituents and was readily hydrolysed under gentle acidic conditions to N-acetylglucosamine 6-phosphate. 3. Studies on the intact polymer showed that it is linear and that adjacent acetamido sugar units are joined by phosphodiester bonds between their 1- and 6-positions, the glycosidic linkages having the α-configuration. This polymer is thus the simplest of the known microbial wall polymers possessing sugar 1-phosphate linkages. 4. Alkali degradation of the extracted polymer proceeds predominantly in a stepwise manner from the reducing end, but evidence was obtained for the direct hydrolysis of some of the inter-unit phosphodiester groups.

The hydrolytic stability of the ester bond of O-aminoacyl derivatives of N-acetylglucosamine, glucosamine (base), and methyl N-acetylneuraminate has been studied at pH 1–8. It has been shown that the stability of the O-aminoacyl derivatives of N-acetylglucosamine is comparable with that of the corresponding glucose derivatives. The presence of a free amino group in glucosamine leads to a considerable labilization of the ester bond. The ester bond of the O-aminoacyl derivatives of methyl N-acetylneuraminate is characterized by a very high lability, and this must be taken into account in working with biopolymers.

We have attempted to detect binding of N-acetylglucosamine (NAG) to alpha-lactalbumin, the B protein of lactose synthetase, under conditions in which binding of NAG to lysozyme, a protein to which alpha-lactalbumin has a significant sequence homology, is observed. Using 1H nuclear magnetic resonance spectroscopy, uv difference spectroscopy, competition of NAG with N-methylnicotinamide chloride, and fluorescence spectroscopy, no binding was detected. The synthesis of a NAG analogue, N-diazoacetyl-glucosamine (diazoNAG), was carried out, and the molecule was demonstrated to be an active galactose acceptor in the lactose synthetase reaction. Use of this molecule in photochemical labeling experiments resulted in a large amount of nonspecific labeling of alpha-lactalbumin, lactose synthetase A protein, ribonuclease, and lysozyme, but competition experiments in the presence of an excess of NAG revealed some specific labeling in the case of A protein and lysozyme, but not with alpha-lactalbumin or a ribonuclease control. Thus, it is highly questionable that a NAG binding site is retained in alpha-lactalbumin; furthermore, it appears that the galacyosyl acceptor makes significant contacts with the A protein rather than alpha-lactalbumin in the lactose synthetase complex.

The binding of 4-(N-acetylaminoglucosyl)-N-acetylglucosamine to lysozyme was studied by both nuclear magnetic resonance (NMR) and temperature-jump methods under comparable conditions. The NMR measurements on the inhibitor spectrum were carried out over a range of inhibitor concentrations including levels at which most of the inhibitor was bound to the enzyme. Data in this region were obtained by a novel difference method in conjunction with correlation spectroscopy. The results from the combination of both experimental techniques demonstrated the existence of a two-step binding mechanism and produced both values for all of the individual rate constants and also the NMR spectral data for the inhibitor in the two enzyme-inhibitor complexes. The later data characterize the environment experienced by the inhibitor at each stage in the binding process and thus provides both a three-dimensional and a dynamic picture of the interaction.

Publisher Summary An ATP-dependent 2-epimerase that catalyzes the interconversion of N -acetylglucosamine and N -acetylmannosamine has been purified from hog kidney. ATP is not absolutely essential for the activity of 2-epimerase, but it exerts a stimulatory effect on the enzyme, causing approximately a 20-fold increase in its activity. The chapter describes the assay method, purification procedure, and properties of N -acetylglucosamine 2-epimerase from hog kidney. The epimerase activity is assayed by measuring the ADP formed in the reaction, by coupling the 2-epimerase and the specific Nacetylglucosamine kinase. The assay consists of: (1) incubation of the 2-epimerase with N-acetylmannosamine (and ATP, if necessary), (2) treatment of the reaction product with an excess of the specific N-acetylglucosamine kinase (ATPase free) and ATP for the complete conversion of any N-acetylglucosamine formed in step 1 into N-acetylglucosamine-6-P and the formation of an equivalent amount of ADP, and (3) the estimation of ADP enzymically by the pyruvate kinase-lactic dehydrogenase assay procedure. All operations of purification procedure are conducted at temperatures between 0° and 4°, and all buffer systems used in the fractionation proeedures contain 1 m M EDTA and 10 m M 2-mercaptoethanol. N-Acetyl-D-glucosamine 2-epimerase activity is demonstrated in the absence of ATP by using high enzyme and substrate concentrations. The ATP-dependent and ATP-independent epimerase activities cannot be separated, and the ratio of the specific activities remains nearly constant throughout the range of 1500-fold purification of the enzyme. The desensitization of 2-epimerase to ATP is brought about by controlled heat and by p -chloromercuribenzoate treatment.

Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends...

A soluble fraction of rat liver converts glucosamine and N -acetylglucosamine in the presence of ATP and UTP to N -acetylneuraminic acid. This system, when supplemented with CTP, forms CMP- N -acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP- N -acetylglucosamine and N -acetylneuraminic acid. Kinetic analysis reveals N -acetylglucosamine 6-phosphate, UDP- N -acetylglucosamine, N -acetylmannosamine, N -acetylmannosamine 6-phosphate and N -acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N -acetylglucosamine to N -acetylmannosamine was seen. Labelled UDP-N-acetylglucosamine, N -acetylneuraminic acid and CMP- N -acetylneuraminic acid, respectively, were obtained in good yield by varying the experimental conditions. A chromatographic method for the separation of N -acetylglucosamine 6-phosphate and N -acetylmannosamine 6-phosphate was developed.

Studies on the enzymatic mechanism of the formation of the N-unsubstituted glucosamine residues in Bacillus cereus cell wall peptidoglycan resulted in finding of an enzyme which hydrolyzes acetamido groups of N-acetylglucosamine residues. The enzyme appears to be specific for undegraded peptidoglycan and is distinguishable from N-acetylglucosamine-6-phosphate deacetylase. It shows a pH optimum of 7.0 and requires Co++ for its full activity.

A method is described for the separation and estimation of a large number of biologically important monosaccharides including hexuronic acids. Procedures are also described for the identification and estimation of disaccharides, trisaccharides, tetrasaccharides and pentasaccharides, and of certain N -acetylglucosamine derivatives such as muramic acid and N -acetylglucosaminylasparagine.

The preparation of [β- 32 P]UDP- N -acetyl[1- 14 C]glucosamine is described. This compound was used in reactions with microsomes of rabbit liver to investigate the pathway of glycoprotein synthesis in this system. The 32 P and 14 C were incorporated into a lipid component while only the 14 C was found in the protein products. The lipid material was purified by ion exchange and two dimensional thin layer chromatography. In both of these systems the 32 P and 14 C migrated together. These results suggested that a single microsomal lipid component contained the β-phosphoryl and N -acetylglucosamine residues of the nucleotide-sugar and therefore UMP had to be the other product in the reaction. The “lipid-sugar” had properties similar to the “lipid intermediates” described for microorganisms. The results presented in this and previous papers suggested that this “lipid-sugar” may serve as an intermediate in glycoprotein synthesis.

The incorporation of N-acetylmannosamine ([3H]ManNAc) and N-acetylglucosamine ([3H]GlcNAc and [14C]GlcNAc) has been studied in rat thyroid hemilobes and homogenates. The radioactive label is incorporated into thyroglobulin (TG), [3H]ManNAc showing a time course more rapid than [3H]GlcNAc. [3H]ManNAc is incorporated directly into 19 S thyroglobulin showing no lower molecular weight-labeled precursors of TG either in the soluble or in the solubilized proteins; [3H]GlcNAc is incorporated into 19 S TG and into precursors both in soluble and solubilized proteins. [3H]ManNAc is converted only into N-acetylneuraminic acid (sialic acid). [3H]GlcNAc and [14C]GlcNAc are incorporated mainly as GlcNAc, and an additional small proportion is converted into sialic acid. Both labeled GlcNAc and sialic acid are found in soluble TG but only GlcNAc is found in membrane-bound TG. [3H]ManNAc is particularly useful to study the last step of carbohydrate incorporation while [3H]GlcNAc is a better tool to study the initiation of carbohydrate incorporation. The presence of the label as sialic acid only in soluble TG suggests that sialic acid is incorporated at the time of TG release from endoplasmic membranes. It may, therefore, serve as an indicator of TG secretion.