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Home / Equine Research Bank / Glycoconj J / Characterisation of the enzymatic 4-O-acetylation of sialic ...
Glycoconjugate journal2001; 17(12); 849-858; doi: 10.1023/a:1010965128335

Characterisation of the enzymatic 4-O-acetylation of sialic acids in microsomes from equine submandibular glands.

Abstract: Microsomes prepared from equine submandibular glands and incubated with tritium-labelled AcCoA incorporated acid-insoluble radioactivity in a manner dependent on time, protein, membrane integrity and AcCoA concentration, with incorporation being optimal at 37 degrees C and pH 6.6. Under the experimental conditions used a K(M) of 32.1 microM for AcCoA and a V(max) of 1.2 pmol/mg protein x min was obtained. The incorporation of acid-insoluble radioactivity was also inhibited by CoA in a competitive manner (K(i)=240 microM), as well as by para-chloromercuribenzoate, 3'-dephospho-CoA, 5'-IDP, 5'-ADP, beta-NAD and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. We demonstrate here that this incorporation of radioactivity into endogenous sialic acid is due to the action of an AcCoA:sialate-4-O-acetyltransferase [EC 2.3.1.44]. Radio thin-layer chromatography analyses of propionic acid-released sialic acids showed that the incorporation of radioactivity correlated with the formation of a radiolabelled species that co-migrated with authentic Neu4,5Ac2. Saponification experiments using NaOH, mouse hepatitis virus strain S and Influenza C/JJ/50 virus also showed that the transfer of [3H]acetyl groups from [3H]AcCoA to endogenous sialic acid acceptors was occurring exclusively at carbon 4 of the pyranose ring.
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Publication Date: 2001-08-21 PubMed ID: 11511809DOI: 10.1023/a:1010965128335Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper investigates the enzymatic process that acetylates sialic acids in horse submandibular gland microsomes. The researchers demonstrated that an enzyme identified as AcCoA:sialate-4-O-acetyltransferase is responsible for this acetylation.

Enzymatic Analysis

  • The researchers began by procuring microsomes from the submandibular glands of horses.
  • They then incubated these microsomes with AcCoA, which was labeled with tritium for tracking purposes.
  • The incorporation of the radioactive AcCoA into the microsomes was assessed under different conditions, such as time, protein content, membrane integrity, and AcCoA concentration.
  • The optimal conditions were determined to be a temperature of 37 degrees Celsius and a pH of 6.6.
  • Under these conditions, a KM (the Michaelis–Menten constant) of 32.1 microM for AcCoA and a Vmax (the maximum rate achieved by the system) of 1.2 pmol/mg protein x min were obtained.

Inhibitory Effects

  • The research also identified inhibitors of the AcCoA incorporation process.
  • CoA was found to inhibit the process in a competitive manner, with a Ki (inhibitory constant) of 240 microM.
  • Several other inhibitors like para-chloromercuribenzoate, 3′-dephospho-CoA, 5′-IDP, 5′-ADP, beta-NAD, and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate were also identified.

Enzyme Identification

  • The research identified the enzyme responsible for the radioactivity incorporation into the endogenous sialic acid to be AcCoA:sialate-4-O-acetyltransferase.
  • Radio thin-layer chromatography was used to analyse the sialic acids released, revealing a radioactive species that matched with Neu4,5Ac2.
  • Saponification experiments also affirmed that the acetyl groups’ transfer from AcCoA to endogenous sialic acid acceptors was taking place only at the number 4 carbon in the pyranose ring.

Cite This Article

APA
Tiralongo J, Schmid H, Thun R, Iwersen M, Schauer R. (2001). Characterisation of the enzymatic 4-O-acetylation of sialic acids in microsomes from equine submandibular glands. Glycoconj J, 17(12), 849-858. https://doi.org/10.1023/a:1010965128335

Publication

Glycoconjugate journal
ISSN: 0282-0080
NlmUniqueID: 8603310
Country: United States
Language: English
Volume: 17
Issue: 12
Pages: 849-858

Researcher Affiliations

Tiralongo, J
  • Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, D-24098 Kiel, Germany.
Schmid, H
    Thun, R
      Iwersen, M
        Schauer, R

          MeSH Terms

          • Acetyl Coenzyme A / metabolism
          • Acetylation
          • Animals
          • Cattle
          • Chromatography, High Pressure Liquid
          • Chromatography, Thin Layer
          • Fluorescence
          • Horses
          • Hydrogen-Ion Concentration
          • Kinetics
          • Microsomes / chemistry
          • Microsomes / enzymology
          • Microsomes / metabolism
          • Radiography
          • Sialic Acids / analysis
          • Sialic Acids / metabolism
          • Submandibular Gland / chemistry
          • Submandibular Gland / enzymology
          • Submandibular Gland / metabolism
          • Temperature

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          Citations

          This article has been cited 9 times.
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