J. Biochem, 1989, Vol. 106, No. 1 143-150
© 1989 Japanese Biochemical Society
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PartialPurification and Characterization of Sialate O- Acetylesterase from Bovine Brain1
Biochemisches Institut, Christian-Albrechts-Universitat Olshausenstr. 40, D-2300 Kiel, F.R.G.
2Present address: Enzomed Biotech, Ansal Chamber-l, 3, Bhikaji Cama Place, New Delhi-110066, India.
From bovine brain an esterase was purified 2, 600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8–6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. tf-naphthyl acetate, the highest preference was for iV-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-9-O-acetylneuraminic acid. Other primary acetyl esters such as 6- O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2, 3-didehydro-iV-acetylneuraminic acid, however, is a substrate for this esterase. Whereas iV-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. iV-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue. Based on its substrate specificity and its preference for sialic acids among the natural substrates, the bovine brain esterase can be classified as sialate O-acetylesterase [EC 3.1.1.53 [EC] ].
1This work was supported by the Fonds der Chemischen Industrie.
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