Characterisation of the enzymatic 4-O-acetylation of sialic acids in microsomes from equine submandibular glands.
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.
Tiralongo
J
J
Biochemisches Institut, Christian-Albrechts-Universität, Olshausenstr. 40, D-24098 Kiel, Germany.
Schmid
H
H
Thun
R
R
Iwersen
M
M
Schauer
R
R
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005453:Fluorescence; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D011859:Radiography; D012794:Sialic Acids / Q000032:analysis / Q000378:metabolism*; D013363:Submandibular Gland / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D013696:Temperature
D012794:Sialic Acids; D000105:Acetyl Coenzyme A