Hexosaminidase assays

Abstract

beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

Ambroxol effects in glucocerebrosidase and α‐synuclein transgenic mice

“…The reaction was stopped by adding 0.25M glycine (pH 10.4), and substrate fluorescence was measured as above 28. All HEXB assays were performed in triplicate.…”

Clinacanthus nutans Aqueous Extract But Not Ethanolic Extract Inhibits the Release of Preformed Mediators in In Vitro Model of IgE-Mediated Mast Cell Degranulation

“…Following that we assessed the inhibitory effect of these extracts on the release of pre-formed mediators of IgE-mediated mast cell degranulation using the non-cytotoxic range of concentrations. Pre-formed mediators such as the lysosomal enzyme -β-hexosaminidase had been reported to be a suitable marker of inflammation as abnormal level of the enzyme has been found in a variety of inflammatory diseases [37] . In addition, it is also a widely accepted marker for mast cell degranulation reaction as it is stored alongside allergic mediators such as histamine and released when mast cells are immunologically stimulated [35] .…”

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…5B to E). Clearly, BbhI shared the common characteristic of all known structures of GH20 enzymes, that is, the (␤/␣) 8 -barrel architecture of the catalytic domain (see Fig. S15 in the supplemental material).…”

“…These enzymes are widely distributed in microorganisms, plants, and animals, and they play crucial roles in nature (6). For instance, they can cleave a GalNAc residue from GM2 gangliosidose, and their deficiency will cause lysosomal storage diseases (7,8). They are also involved in bacterial cell wall recycling, fungal cell wall chitin lysis, and the insect life cycle (9)(10)(11).…”