A modification of determination for glucosamine and galactosamine in glycoprotein with the amino acid analyzer: Application to total acid hydrolyzate of rat renal glomerular basement membrane

A procedure was described for the rapid determination of glucosamine and galactosamine in total acid hydrolyzate of rat renal glomerular basement membrane (GBM) by the use of amino acid analyzer. Glucosamine and galactosamine, as well as other amino acids in glycoprotein hydrolyzate, were well identified and estimated simultaneously by using a short column of HITACHI I-PF-B spherical resin, eluted with a pH 6.09 buffer containing 8% methanol at 38°C.Total time consumed for elution of galactosamine was 60 min. This method is ideal for the separation of small amount of galactosamine from hydroxylysine-rich materials.     

The determination of glucosamine and galactosamine in some glycoproteins by radioisotope dilution

1. The principle of radioisotope dilution was applied on a semi-micro scale to the determination of glucosamine and galactosamine in some glycoproteins, such as immunoglobulins, a urinary glycoprotein and blood-group-specific substances. 2. The glycoprotein was hydrolysed in the presence of [1-(14)C]glucosamine or [1-(14)C]galactosamine or both. The amino sugars were made to react with naphthyl isothiocyanate and the products formed were isolated by the method of Scott (1962). The specific radioactivities determined from liquid-scintillation counting and the extinction at 240mmu or 222mmu were used to calculate the content of amino sugars in the protein analysed. 3. Where the values could be compared with those found by other workers, differences were in general not very great. The advantages of the method are that high concentrations of acid can be employed and undesirable side reactions, which may occur with the free sugars, do not affect the results. A potential source of error of the method is discussed.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.     

东北黑土氨基糖的矿化动态及其对外源物质添加的响应

采用间歇淋洗好气培养法研究了东北黑土中3种不同微生物来源氨基糖(氨基葡萄糖、胞壁酸和氨基半乳糖)的矿化动态以及对葡萄糖添加和葡萄糖与氮肥配施的响应.结果表明:土壤中不同种类的氨基糖具有不同的矿化特征.培养期间胞壁酸含量减少25.4%而氨基葡萄糖含量降低7.1%,表明细菌来源的胞壁酸在土壤中的矿化速率快于真菌来源的氨基葡萄糖,但氨基葡萄糖的矿化数量(68.4 mg·kg^-1)显著高于胞壁酸(15.4 mg·kg^-1).葡萄糖添加以及葡萄糖与氮肥配施均显著提高了土壤中氨基葡萄糖和胞壁酸的含量,但两种处理的影响有所不同.相比之下,氨基半乳糖在土壤中矿化较慢,并且受外源物质的影响较小,表现出较高的稳定性.     

Determination of amino sugars in mixtures containing glucosamine, galactosamine and muramic acid

A colorimetric method is described whereby the direct quantitative determination of glucosamine, galactosamine and muramic acid can be achieved without previous treatment of the cell-wall hydrolysate, for example by column chromatography. Molar ratios of hexosamines in cell-wall preparations, from a number of bacterial species, determined by this method were found to be in general agreement with previously published results.     

Gas chromatographic analysis of hexosamines in glycoproteins

A gas chromatographic method for the analysis of hexosamines in glycoproteins was described which uses the alditol acetate derivatives of the sugars. A polyamide (Poly A 103) liquid phase was used which effectively separates glucosamine, galactosamine, and mannosamine from each other. Mannosamine served as internal standard to facilitate accurate quantitation of glucosamine and galactosamine.     

Enzymatic determination of itoic acid, a Bacillus subtilis siderophore, and 2,3-dihydroxybenzoic acid.

A specific enzymatic method to determine the amounts of itoic acid, a Bacillus subtilis siderophore, and 2,3-dihydroxybenzoic acid (2,3-DHBA) was devised. A sample was incubated first with hippurate hydrolase and then with 2,3-DHBA-3,4-dioxygenase. Itoic acid was estimated from the increase in A374. The incubation with the first enzyme was omitted for the determination of 2,3-DHBA.     

The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of an ascites hepatoma, AH 130 FN.

Plasma membranes were isolated from an ascites hepatoma, AH 130 FN, a free-cell type subline of AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared from the membranes by pronase digestion then fractionated chromatographically and electrophoretically. Isolated fractions were analyzed for amino acid and carbohydrate compositions. The results were compared with those for corresponding fractions from AH 66 and AH 130 ((1974) J. Biochem. 76, 319-333; (1975) ibid., 78, 863-872). The fraction excluded from Sephadex G-50 contained mucopolysaccharides and a series of glycopeptides. The mucopolysaccharides were identified as chondroitin sulfate A on the basis of their chemical composition, electrophoretic behavior on cellulose acetate and digestibility with chondroitinase AC [EC 4.2.2.5]. This contrasts with previous findings that mucopolysaccharides from the corresponding fractions from AH 130 and AH 66 were heparan sulfate. The chemical composition of the glycopeptides, which showed high contents of threonine, serine, galactose, galactosamine, glucosamine, and sialic acid, indicated the presence of glycopeptides with O-glycosidic linkages. The glycopeptides also contained a small but significant amount of aspartic acid, suggesting that N-glycosidic glycopeptides were also contained in this fraction. The fraction included in Sepnadex G-50 contaoned N-glycosidic glycopeptides as major components, since the carbohydrate moieties were composed of fucose, galactose, mannose, glucosamine, sialic acid, and a smaller amount of galactosamine. The presence of galactosamine suggested that O-glycosidic glycopeptides were present as minor components. Glycopeptides with both O- and N-glycosidic linkages were isolated from AH 130, but not from AH 66.     

Synthesis of N3-fumaramoyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase

Several analogues of N3-fumaramoyl-L-2,3-diaminopropanoic acid were synthesized and evaluated for inhibition of glucosamine-6-phosphate synthetase activity. The syntheses were accomplished by acylation reaction of N2-tert.-butoxycarbonyl-L-2,3-diaminopropanoic acid (Boc-A2pr) or N2-tert.-butoxycarbonyl-L-2,4-diaminobutanoic acid (Boc-A2-bu) with the N-succinimidoyl esters of several derivatives of alpha, beta-unsaturated acids 2a-d followed by deprotection of the Boc groups. The obtained compounds were tested for inhibition of glucosamine synthetase isolated from Salmonella typhimurium and Saccharomyces cerevisiae. The results indicated that among the synthesized compounds, N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP) was the most powerful inhibitor of glucosamine synthetase.     

Biochemical study of cardiac valvular tissue. Biosynthesis in vitro of hexosamine-containing substances in bovine heart valve

1. Two distinct hexosamine-containing substances have been obtained from bovine cardiac valvular tissue which was incubated with labelled glucosamine. These were identified as mucopolysaccharides and glycopeptides respectively, both by the elution pattern from a Sephadex G-50 column and by chemical analysis. 2. In the mucopolysaccharide fraction about 80% of both the total hexosamine and total radioactivity were present; the galactosamine/glucosamine ratio of the amounts was about 1.36. 3. The glycopeptide fractions had about 20% of both total hexosamine and total radioactivity; the galactosamine/glucosamine ratio of amounts was about 0.44. In this fraction, over 15% of radioactivity was present in sialic acid. 4. In contrast with the concentrations of the several chemical components, there were remarkable differences in the biosynthetic activities among the four valves; the tricuspid valve had highest specific radioactivity in all components of both substances, followed then in turn by mitral, aortic and pulmonary valves. 5. Glucosamine in mucopolysaccharides had a rapid rate of turnover, followed then in turn by turnover rates of both glucosamine and galactosamine in glycopeptides, and of galactosamine in mucopolysaccharides. The possible significance of these findings is discussed.     

Two-column system for determination of glucosamine, galactosamine, and amino acids on a Beckman 121MB amino acid analyzer: separation of the anomers of glucosamine and galactosamine.

A procedure is described for the determination of glucosamine, galactosamine, and the common amino acids in glycoproteins by the use of a Beckman 121MB amino acid analyzer. The procedure employs Beckman AA10 ion exchange resin in a two-column system. Separation of the hexosamines and their anomers along with the basic amino acids on a short column and of the acidic and neutral amino acids on a long column is achieved. The use of the two-column system permits quantitation of the hexosamines and amino acids in the 100–1000 pmol range, corresponding to 2–20 μg of glycoprotein analyzed. The separation of the α and β anomers of the hexosamines is critically dependent on pH. Galactosamine yields two peaks at pH 6.20 and glucosamine one, and glucosamine yields two peaks at pH 6.74 and galactosamine one. Separation of the anomers improves at lower temperatures (25 versus 40°C) but is relatively insensitive to ionic strength (0.1 to 0.4 n in sodium).     

High-throughput analysis of hexosamine using a colorimetric method

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives—glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride—were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.     

The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of an ascites hepatoma, AH 130.

Plasma membranes were isolated from an ascites hepatoma, AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared by digesting the membranes with pronase, then by fractionating the digest chromatographically and electrophoretically. Isolated fractions were analyzed for their amino acid and carbohydrate compositions. Results were compared with those for corresponding fractions from AH 66 (J. Biochem. 76, 319-333 (1974)). Mucopolysaccharides and a series of glycopeptides were isolated from the fraction excluded from Sephadex G-50. The mucopolysaccharides were identified as a family of heparan sulfates with different electrophoretic mobilities. The glycopeptides contained serine, threonine, galactose, galactosamine, glucosamine, and sialic acid as the major constituents as aspartic acid and mannose as minor ones. This suggests that most of the carbohydrate moieties are linked to serine or threonine (O-glycosidic), and that some are linked to asparagine (N-glycosidic). No nearly purely O-glycosidic glycopeptides were found in this fraction from AH 130, through they were the major glycopeptides from the AH 66 plasma membranes. In the fraction included in the gel, glycopeptides containing fucose, galactose, mannose, glucosamine, glaactosamine, and sialic acid were found. The presence of galactosamine suggests that some of the glycopeptides are O-glycosidic though most are N-glycosidic. In the corresponding fraction from AH 66, nearly purely N-glycosidic glycopeptides were found.     

The identification and quantitation of connective tissue hexosamines using a combination of gas liquid chromatographic and colorimetric procedures.

Due to interactions between amino sugars, amino acids, and/or carbohydrate breakdown products from acid hydrolysis, the quantitation of individual amino sugars from connective tissue hydrolysates, requires a number of indirect steps involving separation and purification of the hexosamines prior to gas-liquid chromatography. In this paper, a method is reported which permits the direct quantitation of galactosamine and glucosamine from connective tissue hydrolysates, utilising a combination of both gas-liquid chromatographic and colorimetric procedures. A two-phase extraction system which selectively eliminates pyridine and amino acids from the T.M.S. ethers of glucosamine and galactosamine is also described.     

The effect of scurvy on hexosamine-containing substances in healing wounds in guinea pigs

1. Granulation tissue from healing tendonectomy wounds in guinea pigs was analysed and the effects of inanition and ascorbic acid deficiency on this tissue were investigated. 2. Inanition produced no significant effect on either the glucosamine or the galactosamine content of the tissue. Ascorbic acid deficiency decreased the galactosamine content without affecting the glucosamine content. 3. Fractionation of papain-digested granulation tissue gave three major fractions, which behaved respectively as glycopeptide, hyaluronic acid and a sulphated glycosaminoglycan mixture. At least half of the sulphated glycosaminoglycan mixture behaved as dermatan sulphate. 4. Inanition produced no consistent effect on the fractions examined. In ascorbic acid deficiency, a decrease in the sulphated glycosaminoglycan fraction was observed, which accounted for the decreased galactosamine content of the tissue. This was accompanied by a decrease in hyaluronic acid and a slight increase in the glycopeptide fraction.     

Grucuronic acid-containing glycopeptide from squid cartilage

A glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its K_av value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 μmol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37°C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.     

Further studies of the chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa

1. Qualitative and quantitative analytical results for the lipopolysaccharide from acetone-dried cells of Pseudomonas aeruginosa (N.C.T.C. 1999) are presented and possible contamination of the material with nucleic acid was further examined. 2. Additional sugars detected (only in large-scale hydrolysates) were mannose and arabinose; traces of spermidine and putrescine were also found. 3. The heptose component is l-glycero-d-mannoheptose. 4. The thiobarbituric acid-positive component is a 3-deoxy-2-octulonic acid, of which only 35-40% links lipid A to the polysaccharide. This linkage is not broken by hydrolysis with acetic acid up to 0.08m. 5. Liberation of lipid A required hydrolysis with 0.1m-hydrochloric acid, which substantially degraded the polysaccharide moiety. 6. Fractions obtained from the degraded polysaccharide by high-voltage electrophoresis were examined; in these, the alanine/galactosamine molar ratio is approx. 1. 7. Hydrazinolysis of whole lipopolysaccharide showed that at least 40% of the alanine is in amide linkage, possibly with galactosamine. 8. Lipid A, solubilized by alkaline methanolysis was fractionated; most of the phosphorus of the higher-molecular-weight fractions was released as P(i) by a phosphomonoesterase. 9. Hydrazinolysis of lipid A destroyed approx. 80% of the glucosamine, and glycosidically linked glucosamine oligosaccharides could not be isolated.     

An Impaired Uptake of Glucosamine in Tunicamycin Resistant Chinese Hamster Ovary Cells

Tunicamycin resistant mutants (TM^R) were isolated and characterized from Chinese hamster ovary cells. One feature of this TM^R mutants was a marked decrease in incorporation of radioactive glucosamine, both into membrane glycoproteins and G protein of vesicular stomatitis virus.

The cellular uptake and incorporation into acid insoluble materials of various radioactive substances, including glucosamine, galactosamine, mannose, 2-deoxyglucose and leucine, was examined for the purpose of determination whether the reduced incorporation of radioactive glucosamine into glycoproteins was due to a defect in the glycosylation step or decreased uptake of glucosamine by cells.

While incorporation of glucosamine and 2-deoxyglucose into acid insoluble fractions was reduced strikingly in the mutants, the incorporation of mannose and leucine were the same as in the parent cells.

The uptake of glucosamine in TM^R cells was lower than that in the wild type cells, and the Km value for glucosamine uptake differed between the mutants and wild type cells. There was no obvious difference in the uptake of 2-deoxyglucose and mannose.     

A single-column amino acid analysis method which resolves hexosamines and several cysteine derivatives

A single-column amino acid analysis method is presented for use in structural studies of glycoproteins. The system gives excellent resolution of glucosamine, galactosamine, cysteic acid, CM-cysteine, AE-cysteine, the internal standard norleucine, and all amino acids normally present in protein hydrolysates.     

A glycopeptide from adenocarcinoma tissue of human lung

A glycopeptide from adenocarcinoma tissue of human lung was extracted by protein digestion with papain (EC 3.4.22.2) and trypsin (EC 3.4.21.4). This component was isolated by Pevikon block electrophoresis. It possessed hexose, glucosamine, fucose, galactosamine, and sialic acid. In its carbohydrate composition and also the abundance of threonin in the peptide moiety it was quite similar to the glycoprotein from gastrointestinal tract. The physical characteristic of the two, such as their optical rotation and viscosity, were also very similar each other.