Characterization of the sialate-7(9)-O-acetyltransferase from the microsomes of human colonic mucosa.
Sialic acids present on human colonic mucins are highly O-acetylated, however, little is known about the underlying enzymatic activity required for O-acetylation in this tissue. Here we report on the substrate specificity, subcellular localization and characterization of the sialate-7(9)-O-acetyltransferase in normal human colonic mucosa. Using CMP-Neu5Ac, the most efficient acceptor substrate of all those tested, the enzymatic activity was found to be optimal at 37 degrees C, with a pH optimum of 7.0. Activity was also found to be dependent on protein, CMP-Neu5Ac (Km: 59.2 microM) and AcCoA (Km: 6.1 microM) concentrations, as well as membrane integrity. The enzyme's activity could be inhibited by CoA with a Ki of 11.9 microM. In addition, enzymatic activity was found to be localized in the Golgi-enriched membrane fraction. The nature of the O-acetylated products formed were verified with the aid of chromatographic and enzymatic techniques. The main product was 9-O-acetylated Neu5Ac, with a significant amount of oligo-O-acetylated Neu5Ac also being detected. The utilization of CMP-Neu5Ac as the acceptor substrate was confirmed by the isolation and characterization of the putative product, CMP-Neu5,9Ac2, using ion-exchange chromatography. The ability of CMP-Neu5,9Ac2 to act as a sialic acid donor for sialyltransferases represents the conclusive demonstration for the formation of CMP-Neu5,9Ac2.
Shen
Yanqin
Y
Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, Germany.
Tiralongo
Joe
J
Iwersen
Matthias
M
Sipos
Bence
B
Kalthoff
Holger
H
Schauer
Roland
R
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003106:Colon / Q000201:enzymology*; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D012794:Sialic Acids / Q000378:metabolism*; D013379:Substrate Specificity
D012794:Sialic Acids; D000123:Acetyltransferases; C018766:N-acylneuraminate-9(7)-O-acetyltransferase