A new and improved assay for endo-β-N-acetylglucosaminidase

✍ Scribed by Anthony L. Tarentino; Frank Maley

• Publisher: Elsevier Science
• Year: 1977
• Tongue: English
• Weight: 596 KB
• Volume: 77
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(77)90304-9

✦ Synopsis

Dansyl-Asn(GlcNAc),3H,* was prepared from Asn(GlcNAc),(Man), by lightly oxidizing with periodate and reducing with sodium borotritide. Following dansylation, the resulting compound was shown to be effective as a substrate for endo+-N-acetylglucosaminidases.

An assay was developed which measures the rate of release of PH](Man),*GlcNAc from dansyl-Asn(GlcNAc),3H,* and is based on the retention of the latter and dansyl-AsnGlcNAc by small columns of AG l-X4 (bicarbonate). The product of the reaction, 3H,*GlcNAc, is recovered by eluting the columns with water and is quantitated in a liquid scintillation spectrometer. The major advantage of this new assay is the absence of the release of radioactivity from dansyl-Asn(GlcNAc),3H,* by exoglycosidases such as cy-D-mannosidase. Unlike previous assays, endo+-N-acetylglucosaminidase activity can be measured reliably in reactions containing tissue extracts as well as purified preparations of the enzyme.