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Biomedical subjects

R Schauer

Publications and source records attributed to R Schauer.

At least 253 records · Page 14Linked to original sources
PubMed1976

The metabolism of O-acyl-N-acylneuraminic acids. Biosynthesis of O-acylated sialic acids in bovine and equine submandibular glands.

1. The enzymic synthesis of 4-O-acetylneuraminic acid, 4-O-acetyl-N-glycolyneuraminic acid, 4-O-glycolyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid and 9-O-acetyl-N-glycolyneuraminic acid is shown using radioactive precursors with surviving slices, membrane fractions or particle-free homogenates from bovine and equine submandibular glands. 2. Acetyl-CoA: N-acetylneuraminate-9(or 7)-O-acetyltransferase activity was found in a microsome fraction and in the cytosol of bovine submandibular glands. The properties of the membrane-bound enzyme acting on endogenous, glycoprotein-bound N-acetyl- and N-glycolylneuraminic acids were compared with those of the soluble enzyme, O-acetylating exogenous, non-glycosidically bound N-acetyl- and N-glycolyneuraminic acids. 3. A rapid, radioactive assay for the membrane-bound enzyme activity is described. The enzyme activity shows an optimum at pH 7 and has a Km for acetyl-CoA of 0.1 mM. The enzyme is inhibited by p-chloromercuribenzoate and iodoacetate. Divalent cations, EDTA and glutathione have no influence on its activity while CoA proved to be a competitive inhibitor with a Ki of 0.56 mM. 4. The soluble enzyme activity, assayed using a radioactive procedure, shows Km values of 0.01 mM, 0.5 mM and 0.39 mM for acetyl-CoA, N-acetylneuraminic acid and N-glycolylneuraminic acid respectively. The general properties are similar to those found for the membrane-bound enzyme, except that membrane-bound activity is stable for longer on storage at 4 degrees C. 5. Acetyl-CoA, acyl-CoA and CoA concentrations of 33 nmol, 65 nmol and 106 nmol/g wet tissue respectively are found in fresh bovine submandibular glands. 6. The occurrence of the CMP-glycosides of N-acetylneuraminic acid, 9-O-acetyl-N-acetyl-neuraminic acid and N-glycolylneuraminic acid in bovine submandibular glands is demonstrated. 7. The results are discussed in relation to the general metabolism of acylneuraminic acids.

Acetyltransferases↗
PubMed1976

N-acetyl-9-O-L-lactylneuraminic acid, a new acylneuraminic acid from bovine submandibular gland.

The acylneuraminic acid fraction, obtained on mild acid hydrolysis of glycoproteins from bovine submandibular glands, contains approximately 2% N-acety-9-O-L-lactylneuraminic acid. The compound has been isolated and purified by ion-exchange and cellulose column chromatography. The structure has been elucidated using thin-layer chromatography, colorimetry, gas-liquid chromatography/mass spectrometry, periodate oxidation and specific lactate dehydrogenases. An evaluation of the different analytical methods is given.

Animals↗
PubMed1976

Neuraminic acid-specific modification and tritium labelling of gangliosides.

1. A crude ganglioside mixture and pure GM1 and GD1a from bovine brain grey matter were prepared on a large scale. 2. The C7- and G8-analogues of NeuNAc were prepared from Collocalia mucoid and their structures established by gas-liquid chromatography and mass spectrometry. 3. Using model compounds in addition to various gangliosides, the conditions for the periodate oxidation and subsequent borohydride reduction of gangliosides were investigated with regard to the yield of C7- and C8-analogues of NeuNAc and the integrity of other monosaccharides in the oligosaccharide chain. These conditions were optimised to yield maximum C8-NeuNAc production and low C7-NeuNAc formation. Thus products were obtained which closely resemble the native gangliosides. 4. Using boro [3H] hydride, ganglioside derivatives with high specific radioactivity were prepared for the first time, containing either NeuNAc and labelled C8-NeuNAc or mainly labelled C7-NeuNAc depending on the prevailing conditions.

Animals↗
PubMed1976

Demonstration of neuraminidase activity in human blood serum and human milk using a modified, radioactively labelled alpha1-glycoprotein as substrate.

N-Acetylneuramini acid of alpha1-glycoprotein was oxidized with a small molar excess of periodate and reduced with tritium-labelled borohydride. By this method about 50% of the N-acetylneuraminic acid was converted to its radioactively labelled C8-analog and 25% to its C7-analog. Using this modified alpha1-glycoprotein as substrate, minimum neuraminidase concentrations of 10(-18) units/ml, related to the activity of neuraminidase from Vibrio cholerae, could be determined. Neuraminidase activity was demonstrated in 95% of the sera or blood plasma samples from a series of 417 healthy or ill human individuals and in milk samples from 5 different donors. The neuraminidase in both serum and milk had optimal activity at pH 5.5. On an average, 10(-10) neuraminidase units were found in 1 ml serum and 10(-8) units in 1 ml milk. Although the neuraminidase activities in the sera varied, a correlation with definite pathological states is not yet possible. N-Acetylneuraminate pyruvate-lyase activity could not be detected in human serum.

Female↗
PubMed1976

Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.

Binding Sites↗
PubMed1976

Neuraminic acid derivatives newly discovered in humans: N-acetyl-9-O-L-lactoylneuraminic acid, N,9-O-Diacetylneuraminic acid and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid.

The free and glycosidically bound acylneuraminic acids from human serum and saliva and the free acylneuraminic acids from human urine have been characterized by thin-layer chromatography and gas-liquid chromatography/mass spectrometry. Acylneuraminic acid mixtures obtained from serum and saliva contain mainly N-acetylneuraminic acid and N-acetyl-9-O-L-lactoylneuraminic acid, whereas small amounts of N,9-O-diacetylneuraminic acid are also present. No free N,O-diacylneuraminic acids could be detected in the urine samples. None of the investigated fluids contained N-glycoloylneuraminic acid. The unsaturated N-acetyl-2,3-dehydro-2-deoxyneuraminic acid is usually a component of the free acylneuraminic acid fractions of serum, saliva and urine. The body fluids of a patient with sialuria contain the same O-acylated and unsaturated N-acetyl neuraminic acid derivatives as mentioned above, but the total amounts of free acylneuraminic acids in these materials are significantly higher than found for normal persons.

Chromatography, Gas↗
PubMed1975

Synthesis of 9-O-acetyl- and 4,9-di-O-acetyl derivatives of the methyl ester of N-acetyl-beta-D-neuraminic acid methylglycoside. Their use as models in periodate oxidation studies.

Reaction of the methyl ester of N-acetyl-beta-D-neuraminic acid methyl glycoside with N-acetylimidazole yielded the corresponding 9-O-acetyl- and 4,9-di-O-acetyl derivatives. The structures of these compounds were confirmed by mass spectrometry and both 1H and 13C NMR spectroscopy. The compounds served as model substances in a comparative study of the rate of periodate oxidation of unsubstituted and of 9-O-acetylated-N-acetyl-neuraminic acids. This reaction was strongly hampered by the presence of the 9-O-acetyl group. The low molar absorbancy coefficient of N-acetyl-9-O-acetylneuraminic acid in the periodic acid/thiobarbituric acid assay can be explained by this retardation.

Chemical Phenomena↗
PubMed1975

A micro-method for quantitative determination of acylneuraminic acids from erythrocyte membranes.

A micro-method is presented which enables the fast and exact determination of acid-hydrolyzed acylneuraminic acids in erythrocyte membranes. Erythrocytes from 1 ml of human and rabbit blood containing ACD buffer are, washed and hemolyzed on Millipore filters of pore size 1.2 mu. Acylneuraminic acids are released from the erythrocyte membranes still on the filters under the optimal conditions of 0.1 N HCl at 80 degrees C for 50 min. A prerequisite for the determination of the true amount of acylneuraminic acids using the periodic acid/thiobarbituric acid assay is the small-scale extraction of lipids from the hydrolysate and anion-exchange chromatography of acylneuraminic acids. The values thus obtained must be corrected, as 20% of acylneuraminic acids are destroyed during acid hydrolysis. In samples of human blood from 10 healthy individuals, on an average 223 nmol acylneuraminic acids per ml of packed erythrocytes were found, and in the same amount of rabbit erythrocytes, 1e method for a screening of the acylneuraminic acid content of erythrocyte membranes in hemolytic diseases or of other cell membranes is discussed.

Animals↗
PubMed1975

Purification and characterization of neuraminidase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.

Clostridium perfringens↗