Partial purification and characterization of sialate O-acetylesterase from bovine brain.
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. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-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-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-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. N-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Schauer
R
R
Biochemisches Institut, Christian-Albrechts-Universität, Kiel, F.R.G.
Reuter
G
G
Stoll
S
S
Shukla
A K
AK
D000115:Acetylesterase; D000818:Animals; D000837:Anion Exchange Resins; D001921:Brain / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007525:Isoelectric Focusing; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight
D000837:Anion Exchange Resins; D002265:Carboxylic Ester Hydrolases; D000115:Acetylesterase; C046298:sialate O-acetylesterase; D008628:Mercury