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Y. Shen, J. Tiralongo, M. Iwersen, B. Sipos, H. Kalthoff, R. Schauer
Characterization of the Sialate-7(9)-O-Acetyltransferase from the Microsomes of Human Colonic Mucosa
Sialic acids present on human colonic mucins are highly Oacetylated, however, little is known about the underlying enzymatic activity required for Oacetylation in this tissue. Here we report on the substrate specificity, subcellular localization and characterization of the sialate-7(9)Oacetyltransferase in normal human colonic mucosa. Using CMPNeu5Ac, the most efficient acceptor substrate of all those tested, the enzymatic activity was found to be optimal at 37 C, with a pH optimum of 7.0. Activity was also found to be dependent on protein, CMPNeu5Ac (Km: 59.2 M) and AcCoA (Km: 6.1 M) concentrations, as well as membrane integrity. The enzymes activity could be inhibited by CoA with a Ki of 11.9 M. In addition, enzymatic activity was found to be localized in the Golgienriched membrane fraction. The nature of the Oacetylated products formed were verified with the aid of chromatographic and enzymatic techniques. The main product was 9-Oacetylated Neu5Ac, with a significant amount of oligoOacetylated Neu5Ac also being detected. The utilization of CMPNeu5Ac as the acceptor substrate was confirmed by the isolation and characterization of the putative product, CMPNeu5,9Ac2, using ionexchange chromatography. The ability of CMPNeu5,9Ac2 to act as a sialic acid donor for sialyltransferases represents the conclusive demonstration for the formation of CMPNeu5,9Ac2.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 383, 02/2002
Pages: 307 - 317