Biochemical and genetic evidence for distinct membrane-bound and cytosolic sialic acid O-acetyl-esterases: serine-active-site enzymes.
A cytosolic sialic acid-specific O-acetyl-esterase was previously described that can remove O-acetyl esters from the 9-position of sialic acids. We show that rat liver Golgi vesicles contain a distinct sialic acid-esterase located within the lumen of the same vesicles that add O-acetyl esters to sialic acids. Studies of a retinoblastoma cell line genetically deficient in the cytosolic enzyme also confirm the existence of distinct membrane-associated sialic acid esterase activity. We developed a sensitive, specific and facile assay, which measures release of [3H]acetyl groups from [3H-acetyl]9-O-acetyl-N-acetylneuraminic acid. Using this assay, we show that rat liver membranes may contain different sialic acid O-acetyl-esterases. The membrane-associated enzyme(s) bind to Concanavalin A Sepharose, whereas the cytosolic enzyme does not. Membrane-bound and cytosolic esterases are inactivated by di-isopropyl-fluorophosphate, showing they are serine-active-site enzymes.
D000115:Acetylesterase; D000818:Animals; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D005134:Eye Neoplasms; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051381:Rats; D012175:Retinoblastoma; D012694:Serine
D007527:Isoenzymes; D012694:Serine; D002265:Carboxylic Ester Hydrolases; D000115:Acetylesterase; C046298:sialate O-acetylesterase