N-acetylneuraminate Pyruvate-lyase Research Articles

Complete nucleotide sequence of the E. coli N-acetylneuraminate lyase.

The nucleotide sequence of the cloned DNA, 1,243 bp in length coding for N-acetylneuraminate lyase (N-acetylneuraminate pyruvate lyase; NPL) of Escherichia coli has been determined. Nucleotide sequence and amino acid analysis have assigned the open reading frame for NPL, starting with the ATG near its 5'terminus. The molecular weight calculated from the predicted amino acid sequence was 32,640 daltons, being in good agreement with that of a NPL subunit estimated by the SDS-PAGE method and amino acid composition. Several signal sequences conserved in the promoter regions of E. coli were found in the npl gene. They were the Shine-Dalgarno sequence, the Pribnow box and the sequence coserved in the "-35 region" and they were separated to each other with preferable spacing for an efficient transcription. Downstream from the termination codon, the inverted repeat sequence was present, followed by 4 successive T's.

Common conditions for high-performance liquid chromatographic microdetermination of aldoses, hexosamines, and sialic acids in glycoproteins

All aldoses known to be present in animal and plant glycoproteins were separated in partition mode on a column of a highly crosslinked cation-exchange resin (Shodex DC-613, H + form) and sensitively monitored by photometric detection after postcolumn labeling with 2-cyanoacetamide. Linearity for the sample amount ranging from 0.1 to 80 nmol was observed with high reproducibility. Hexosamines could be determined as their N-acetyl derivatives under the identical conditions with approximately one order of magnitude higher range of linearity. Sialic acid could be estimated as N-acylhexosamines produced by the action of N-acetylneuraminate pyruvate-lyase. The usefulness of these common conditions was demonstrated by analyzing component monosaccharides of some glycoprotein preparations.

Preparation of 9-fluoro-9-deoxy- N-[2- 14C]acetylneuraminic acid : Activation and transfer onto asialo-α 1-acid glycoprotein

9-Fluoro-9-deoxy- N-[2- 14C]acetylneuraminic acid has been prepared from 6-fluoro-6-deoxy- N-acetylmannosamine and [2- 14C]pyruvic acid for the first time, using Clostridium perfringens N-acetylneuraminate pyruvate-lyase (EC 4.1.3.3). The fluoro sugar was activated by CMP- N-acetylneuraminic acid synthase and CTP to yield CMP-9-fluoro-9-deoxy- N-[2- 14C]acetylneuraminic acid. Both products were obtained in good yield (60 and 30%, respectively). The radioactive sugar in its activated form is glycosidically attached to asialo-α 1-acid glycoprotein by sialyltransferase and can be removed by the action of Vibrio cholerae sialidase. The reaction rates of the enzymes studied are lower with the 9-fluoro derivatives than with the N-acetylneuraminic acid substrates.

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.

Neuraminidase and N-acetylneuraminate pyruvate-lyase of Pasteurella multocida.

SUMMARY Neuraminidase was found in nearly all strains of Pasteurella multocida, in some strains of P. haemolytica, but not in P. pseudotuberculosis. The enzyme of P. multocida is bound to the bacterial cell. It is inducible by N-acetyl-D-mannosamine and by free and bound sialic acid. After solubilization it has a molecular weight of about 250000, a pH optimum = 6.0, and a Michaelis constant Km = 2 ⨯ 10-4 M when 3’-sialyllactose is used as substrate. N-acetylneuraminate pyruvate-lyase activity present in Pasteurella multocida is higher in the cell-free culture medium than in the bacterium. It is suggested that the stimulating effect of blood sera on the production of neuraminidase by Pasteurella multocida is due to the delayed release of the inducer N-acetyl-D-mannosamine.