Solubilisation and properties of the sialate-4-O-acetyltransferase from guinea pig liver.
The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate-4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme O-acetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble O-acetyltransferase maximally operated at 30 degrees C, pH 5.6, and 50-70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 microM for AcCoA and 1.2 microM for GD3. CoA inhibits the enzyme with a Ki value of 14.8 microM. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration.
Iwersen
Matthias
M
Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, D-24098 Kiel, Germany.
Dora
Hiltrud
H
Kohla
Guido
G
Gasa
Shinsei
S
Schauer
Roland
R
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002793:Cholic Acids; D002855:Chromatography, Thin Layer; D005732:Gangliosides / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D006168:Guinea Pigs; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D012799:Sialyltransferases / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D014462:Ultrafiltration
D002793:Cholic Acids; D005732:Gangliosides; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C118584:N-acetylneuraminate 4-O-acetyltransferase; D012799:Sialyltransferases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate