THE LYSOSOME PERIPHERY: BIOCHEMICAL AND ELECTROKINETIC PROPERTIES OF THE TRITOSOME SURFACE

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO3, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm2, and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 µg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 ± 3; glucosamine, 24 ± 3; galactosamine, 10 ± 2; glucose, 21 ± 2; galactose, 26 ± 2; mannose, 31 ± 5; fucose, 7 ± 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.

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Investigations on the oligosaccharide units of an A myeloma globulin

Biochemical Journal ◽

10.1042/bj1070341 ◽

1968 ◽

Vol 107 (3) ◽

pp. 341-352 ◽

Cited By ~ 84

Author(s):

G. Dawson ◽

J. R. Clamp

Keyword(s):

Sialic Acid ◽

Electrophoretic Mobility ◽

Acid Content ◽

Carbohydrate Content ◽

Glycosidic Linkage ◽

Core Oligosaccharide ◽

Sialic Acid Content ◽

Acetylneuraminic Acid ◽

The Core ◽

Polypeptide Chains

The carbohydrate content of an A myeloma globulin was investigated. The carbohydrate content was found to be unchanged when the protein was isolated from the patient over a period of 18 months. The various polymeric forms of the protein contained similar proportions of carbohydrate. The A myeloma globulin contained approx. 2 residues of 6-deoxy-l-galactose (l-fucose), 14–15 of d-mannose, 12–13 of d-galactose, 12–13 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine), 6 of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) and 5 of N-acetylneuraminic acid (sialic acid), and these were distributed between six oligosaccharide units all of which were present on the heavy polypeptide chains. The oligosaccharide units showed two kinds of heterogeneity, which have been termed central and peripheral. Central heterogeneity was shown by the presence of three completely different core units, which had the following compositions: (1) 3 residues of d-galactose and 3 of 2-acetamido-2-deoxy-d-galactose, joined to protein by an O-glycosidic linkage between acetamidohexose and serine; (2) 3 residues of d-mannose, 2 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, joined to protein by an N-glycosidic linkage between acetamidohexose and aspartic acid; (3) 4 residues of d-mannose and 3 of 2-acetamido-2-deoxy-d-glucose with a linkage similar to that in (2). The core oligosaccharide units showed peripheral heterogeneity in the attachment of 6-deoxy-l-galactose, 2-acetamido-2-deoxy-d-glucose and N-acetylneuraminic acid. Tentative structures are proposed for these various types of oligosaccharide unit. Glycopeptides were isolated in which the sialic acid content exceeded that of d-galactose. Explanations are given for the electrophoretic mobility and staining characteristics of the various glycopeptides.

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α2-Adrenoreceptor Status of Human Platelet Subpopulations Separated by Continuous Flow Electrophoresis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651549 ◽

1993 ◽

Vol 69 (01) ◽

pp. 060-063 ◽

Cited By ~ 3

Author(s):

M Crook ◽

N Crawford

Keyword(s):

Sialic Acid ◽

Electrophoretic Mobility ◽

Continuous Flow ◽

Human Platelet ◽

Human Platelets ◽

T Test ◽

Total Sialic Acid ◽

Student’S T ◽

Student’S T Test

SummaryHuman platelets were separated into subpopulations using continuous flow electrophoresis, which uses electrophoretic mobility as the separative parameter. The platelets with the greatest electrophoretic mobility showed higher amounts of total sialic acid than the less electrophoretically mobile subpopulation (74.3 ± 18.2 nmol/109 platelets vs. 49.2 ± 20.1 nmol/109 platelets, p <0.05 paired Student’s t-test). Furthermore, neuraminidaselabile sialic acid was also elevated in the more electrophoretically mobile platelet subpopulation (29.1 ± 6.0 nmol/109 platelets vs. 21.8 ± 10.4 nmol/109 platelets, p <0.05 paired Student’s t-test). We also found that the sialic acid enriched platelet subpopulation had more α2-adrenoreceptors than the less electrophoretically mobile platelets (457 ± 104 vs. 302 ± 164 receptors per cell, p <0.05 paired Student’s t-test).

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Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system

Biochemical Journal ◽

10.1042/bj2460287 ◽

1987 ◽

Vol 246 (2) ◽

pp. 287-294 ◽

Cited By ~ 14

Author(s):

L B Rodríguez-Aparicio ◽

A Reglero ◽

J M Luengo

Keyword(s):

Escherichia Coli ◽

Sialic Acid ◽

Transport System ◽

Biochemical Characterization ◽

Osmotic Shock ◽

Metabolic Inhibitors ◽

Acid Uptake ◽

Uptake System ◽

Acid Transport ◽

Acetylneuraminic Acid

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.

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The carbohydrate components of hydrolysates of gastric secretion and extracts from mucous glands of the gastric body mucosa and antrum

Biochemical Journal ◽

10.1042/bj1060523 ◽

1968 ◽

Vol 106 (2) ◽

pp. 523-529 ◽

Cited By ~ 33

Author(s):

J. Schrager ◽

M. D. G. Oates

Keyword(s):

Liquid Chromatography ◽

Sialic Acid ◽

Gastric Secretion ◽

The Body ◽

Gastric Body ◽

Amino Sugars ◽

Gas Liquid Chromatography ◽

Acetylneuraminic Acid ◽

Carbohydrate Components

1. The sugars and amino sugars of hydrolysates of gastric secretion were determined by gas–liquid chromatography. 2. All the gastric aspirations examined showed on hydrolysis the presence of fucose, galactose, mannose, glucose, galactosamine, glucosamine, N-acetylneuraminic acid and sulphate. 3. Galactose and glucosamine were always found in equimolar amounts, but the galactose/galactosamine ratio in different aspirations was 2:1, 3:1, 4:1 or 5:1. Repeated gastric aspirations of each subject examined showed constant ratios of these carbohydrate components. 4. Fucose and sialic acid appear to be related to glucosamine and galactosamine respectively. 5. The carbohydrate components of extracts from the mucous glands of the body mucosa and antrum did not differ from those of gastric secretion.

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Regulation of N-glycolylneuraminic acid biosynthesis in developing pig small intestine

Biochemical Journal ◽

10.1042/bj20021049 ◽

2003 ◽

Vol 370 (2) ◽

pp. 601-607 ◽

Cited By ~ 17

Author(s):

Yanina N. MALYKH ◽

Timothy P. KING ◽

Elizabeth LOGAN ◽

Denise KELLY ◽

Roland SCHAUER ◽

...

Keyword(s):

Small Intestine ◽

Sialic Acid ◽

Post Partum ◽

Acid Biosynthesis ◽

Acetylneuraminic Acid ◽

Total Sialic Acid ◽

Key Factor ◽

Developmental Phases ◽

Cytosolic Factors

N-Glycolylneuraminic acid (Neu5Gc), an abundant sialic acid in animal glycoconjugates, is formed by the enzyme CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. The amount of Neu5Gc relative to other sialic acids is highly dependent on the species, tissue and developmental stage. Although the activity of the hydroxylase is a key factor in controlling Neu5Gc incorporation in adult animals, little is known about the regulation of hydroxylase expression and the role of this enzyme in determining changes in Neu5Gc during development. Using pig small intestine as a model system, the appearance of total sialic acid and the regulation of Neu5Gc biosynthesis during development were studied in various regions of this tissue. The amount of total sialic acid and Neu5Gc declined markedly in 2 weeks after birth. Although in subsequent developmental phases there were no positional differences in total sialic acid, a significant proximal-to-distal increase in Neu5Gc was detected. In all cases, a good correlation between the amount of Neu5Gc, the activity of the hydroxylase and the level of hydroxylase mRNA was observed. However, Western-blot analysis revealed considerable accumulation of less active enzyme in the post partum period, which persisted until adulthood. No evidence for cytosolic factors influencing the hydroxylase activity or for the formation of truncated enzyme was found, raising the possibility that other regulatory mechanisms are involved. The relevance of these results in the formation of Neu5Gc as a receptor for certain pig enteric pathogens is also discussed.

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Sialoglycoproteins and sialic acids ofPlasmodium knowlesischizont-infected erythrocytes and normal rhesus monkey erythrocytes

Parasitology ◽

10.1017/s0031182000065422 ◽

1986 ◽

Vol 92 (3) ◽

pp. 527-543 ◽

Cited By ~ 7

Author(s):

R. J. Howard ◽

G. Reuter ◽

J. W. Barnwell ◽

R. Schauer

Keyword(s):

Sialic Acid ◽

Rhesus Monkey ◽

Acid Content ◽

Sialic Acids ◽

Galactose Oxidase ◽

Parasite Development ◽

Sialic Acid Content ◽

Acetylneuraminic Acid ◽

Total Sialic Acid ◽

Significant Difference

SUMMARYThe effects of malaria infection on RBC sialic acids and sialoglycoproteins were studied with asexual blood-stage infections ofPlasmodium knowlesiin rhesus monkeys. Glycoprotein radio-isotope labelling methods were used to compare the sialoglycoproteins of normal RBC andP. knowlesischizont-infected RBC (SI-RBC). Tritiation of glycoproteins from SI-RBC with the standard sialidase + galactose oxidase/NaB3H4method or standard periodate/NaB3H4method was significantly decreased when compared to normal RBC. However, tritium uptake into glycoproteins was normal when SI-RBC were treated with 5-fold higher concentrations of both enzymes in the first labelling method, or with a 5-fold increase in the molar ratio of periodate to sialic acid in the second method. The mobility of tritiated host cell glycoproteins on SDS–polyacrylamide gels was identical for SI-RBC and normal RBC. New bands, possibly glycoproteins, of 230, 160, 90, 52, and 30 kDa were detected after labelling SI-RBC by the modified periodate/NaB3H4method. Sialic acid analysis of normal rhesus monkey RBC (62μg/1010RBC) revealed that 46% of the total sialic acid wasN-glycolylneuraminic acid, 33% wasN-acetyl-9-O-acetylneuraminic acid, and the remainderN-acetylneuraminic acid. SI-RBC collected either directly from infected monkeys or afterin vitroculture of ring-infected RBC in horse serum, had increased total sialic acid (126 or 115μg/1010RBC, respectively). The sialic acid content of infected RBC must increase during parasite development since RBC infected with ring-stageP. knowlesihad the same content as normal RBC. There was no significant difference in the ratio of the three sialic acids of SI-RBC and normal RBC. In contrast, the uninfected RBC from infected blood of different monkeys showed marked variation in sialic acid composition and generally had a lower sialic acid content than normal RBC.

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Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

Biochemical Journal ◽

10.1042/bj1240591 ◽

1971 ◽

Vol 124 (3) ◽

pp. 591-604 ◽

Cited By ~ 45

Author(s):

M. Isemura ◽

K. Schmid

Keyword(s):

Sialic Acid ◽

Acid Hydrolysis ◽

Alkaline Hydrolysis ◽

Nitrous Acid ◽

Rapid Determination ◽

Carbohydrate Moiety ◽

Gas Liquid Chromatography ◽

Acid Glycoprotein ◽

Acetylneuraminic Acid ◽

First Time

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma α1-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1→4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2→2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl→N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas–liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.

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Sialic Acids in Human Gastric Aspirates: Detection of 9-O-Lactyl- and 9-O-Acetyl-N-Acetylneuraminic Acids and a Decrease in Total Sialic Acid Concentration with Age

Clinical Science ◽

10.1042/cs0840573 ◽

1993 ◽

Vol 84 (5) ◽

pp. 573-579 ◽

Cited By ~ 26

Author(s):

A. P. Corfield ◽

S. A. Wagner ◽

A. Safe ◽

R. A. Mountford ◽

J. R. Clamp ◽

...

Keyword(s):

Sialic Acid ◽

Thin Layer ◽

Acid Content ◽

Colorimetric Assay ◽

Sialic Acids ◽

Gas Chromatography Mass Spectrometry ◽

Sialic Acid Content ◽

Acetylneuraminic Acid ◽

Total Sialic Acid ◽

Thin Layer Chromatographic Analysis

1. The total sialic acid content of human gastric aspirates was measured using a colorimetric assay. Care was taken to optimize the assay and to eliminate interference. 2. The sialic acid content of gastric aspirates collected under resting conditions from 77 patients with non-ulcer dyspepsia was found to decrease with age from >100 μg/ml at 25 years and younger to <20 μg/ml above 70 years of age. 3. Analysis of the sialic acids by gas chromatography, mass spectrometry and thin-layer chromatography showed the presence of N-acetylneuraminic acid and two O-acylated derivatives, 9-O-acetyl- and 9-O-lactyl-N-acetylneuraminic acids. These forms were predominantly glycosidically bound. 4. Thin-layer chromatographic analysis of individual aspirate samples showed that the O-acetylated sialic acids were present in all samples, with a maximum of 25% and a minimum of 5% of the total sialic acids.

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