A sialidase from horse liver was co-purified with b-galactosidase and carboxypeptidase A

Abstract

Sialidase (EC 3.2.1.18) is a hydrolytic enzyme that releases sialic acids, which are usually bound through ?-2,3, ?-2,6, or ?-2,8 linkages to oligosaccharides, glycoproteins or glycolipids. In mammals sialidases have been found in various cellular locations, either as cytosolic or membrane-bound enzymes (e.g. lysosomes, Golgi, plasma membrane and nuclear membrane) [1]. The study of membrane-bound sialidases is fraught with difficulties, due to their low stability and their occurrence in a complex with other enzymes such as acid ?-galactosidase and carboxypeptidase A [2, 3, 4, 5]. Sialic acids comprise a family of about 40 different derivatives of the 9-carbon sugar neuraminic acid. Sialic acids with a 4-O-acetyl group occur in a number of mammals, including horse, donkey, guinea pig as well as in the monotreme Echidna [1]. Sialic acids containing this modification are resistant to most sialidases [6]. Although the catabolism of 4-O-acetylated neuraminic acid derivatives in horse liver involves the action of an esterase [7], studies on the sialidase from this tissue may provide further insight into the degradation of sialylated glycoconjugates and enhance our understanding of eukaryotic sialidases. Here we show that the sialidase from horse liver is a membrane-bound enzyme. The solubilized enzyme was isolated using Fractogel TMAE (650) followed by affinity chromatography on the ?-galactosidase- specific medium p-aminophenylthio-?-D-galactopyranoside-agarose and chromatofocusing on PBE 94. The enzyme was found to occur in a complex with ?-galactosidase and carboxypeptidase A. Using sialyl-methylumbelliferyl ?-glycoside as substrate the isolated sialidase exhibits temperature and pH optima of 42-46°C and 4.5 respectively, while the ?-galactosidase is optimally active at 50°C and pH 4.0 with galactosyl-methylumbelliferyl ?-glycoside. On the other hand, the carboxypeptidase A shows temperature and pH optima of 42°C and 5.8, respectively, with N-Carbobenzyloxy-Phe-Leu.