O-GlcNAcase, also known as N-acetylglucosaminidase or hexosaminidase C (EC 126.96.36.199), is a cytosolic glycosidase specific for O-linked ^-GlcNAc. The activity of O-GlcNAcase can be conveniently assayed in vitro with a synthetic substrate, p-nitrophe-nol N-acetylglucosaminide (pNP-^-GlcNAc). The cleavage product, pNP, has an absor-bance peak at 400 nm.
Partially purified O-GlcNAcase (0.2 to 1 |g) or cell extract sample (20 to 50 |g, precipitated with 30% to 50% ammonium chloride) 10x O-GlcNAcase assay buffer (see recipe)
100 mM (50x) p-nitrophenol N-acetylglucosaminide (pNP-GlcNAc) in DMSO 500 mM Na2CO3
96-well flat bottom plates or 1.5 ml microcentrifuge tubes Plate reader or spectrophotometer
Native or recombinant O-GlcNAcase can be partially purified from animal tissues or cultured cells by several chromatographic steps (Dong and Hart, 1994; Gao et al., 2001). Alternatively, a crude enzyme preparation can be generated by passing cell extract over a 1-ml Con-A column. Most of the interfering acidic hexosaminidases are modified by N-linked sugars and bind to Con-A, while neutral O-GlcNAcase is in the flow-through (Izumi and Suzuki, 1983).
2. Precool 96-well plate or microcentrifuge tubes on ice.
3. Set up reactions in the precooled plate wells or tubes as follows:
1 to 50 |l partially purified O-GlcNAcase enzyme or cell extract 10 |l 10x O-GlcNAcase assay buffer
The total reaction volume can be scaled up to 500 jul in microcentrifuge tubes.
pNP-GlcNAc breaks down chemically. A blank reaction without enzyme should be included to determine the background.
50 mM GalNAc is included in the reaction to inhibit lysosomal hexosaminidases A and B which may be present in the enzyme preparation. O-GlcNAcase is not inhibited by 50 mM GalNAc.
4. Mix well and cover.
Yellow color will develop as pNP-GlcNAc is hydrolyzed by O-GlcNAcase. Reactions should be optimized to keep the absorbance within the linear range of the spectrophotometer. The authors find that 20 to 50 ug of cell extract used in a reaction of 100 ul, with a 1 to 2 hr incubation time is appropriate.
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