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Endo-ß-N-acetylglucosaminidase Acting on Carbohydrate Moieties of Glycoproteins

PURIFICATION AND PROPERTIES OF THE ENZYME FROM DIPLOCOCCUS PNEUMONIAE

From the ¹ From the Department of Biochemistry, Kobe University School of Medicine, Kusunoki-cho, Ikuta-ku, Kobe, Japan

A purified ovalbumin glycopeptide, Asn-(GlcNAc)₂(Man)₅ was [¹⁴C]acetylated in the asparagine moiety. Using the [¹⁴C]acetylated glycopeptide as a substrate, an endo-ß-N-acetylglucosaminidase was purified 2400-fold from the culture fluid of Diplococcus pneumoniae. The enzyme preparation was practically free from various exoglycosidases and proteases. The enzyme cleaved the di-N-acetylchitobiose structure of the [¹⁴C]acetylated glycopeptide, yielding equimolar amounts of [¹⁴C]acetyl-Asn-GlcNAc and (Man)₅(GlcNAc). The enzyme had a pH optimum of 6.5, with a K_m of 0.25 mm and with a V_max of 4.67 µmoles per mg of protein per min toward the substrate.

A mouse myeloma IgG glycopeptide and a bovine IgG glycopeptide was hydrolyzed by the enzyme, only after treatment with ß-galactosidase and ß-N-acetylglucosaminidase. In both cases the products of the enzymic action were a fucose-containing glycopeptide fragment and a mannose-containing oligosaccharide whose reducing end was N-acetylglucosamine.

The enzyme had a strict specificity with respect to the structures of oligomannosyl cores of glycopeptides. Thus, the enzyme hydrolyzed only 20% of glycopeptide mixtures from ovalbumin. Furthermore, the enzyme hydrolyzed porcine thyroglobulin glycopeptides, Unit B (sialic acid containing heteropolysaccharide unit) which had been treated with neuraminidase, ß-galactosidase, and ß-N-acetylglucosaminidase, but did not hydrolyze calf thyroglobulin glycopeptides, Unit A (mannose-N-acetylglucosamine unit).

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