ture are incubated with the colorimetric substrate p-nitrophenyl-a-D-N-acetylglucosaminide. At pH 10, the product of hydrolysis, nitrophenyl, changes color and can be quantified spectrophotometrically at 420 nm. The intensity of absorbance, normalized to the total protein Oleandrin web content, correlates with the concentration of metabolized substrate and is thus an indication of enzymatic activity. The fluorogenic assays is based on the use of 4-Methylumbelliferyl-2-acetamido-2-deoxyalpha-D-glucopyranoside, a substrate that releases fluorescent 4-methylumbelliferone upon NAG-mediated cleavage of glycoside 1 and that has advantages in sensitivity and ease of use over the colorimetric substrate. However, similar to the chromogenic assay, this method requires large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content. Alternative and more laborious assays to measure enzyme activities in cultured fibroblasts from MPS III patients include affinity capture2release purification of biotin-tagged products followed by electrospray mass spectrometry and 
the use of radiolabelled oligosaccharides as substrate followed by the measure of the released radioactivity to quantify NAG activity. In an attempt to develop an assay amenable to high-throughput applications we developed and validated a rapid and sensitive method to quantify lysosomal NAG activity based on the use of MUG. All stepsfrom cell treatment with a candidate therapeutic agent to assessment of NAG activitycan be carried out in a 96well plate, making this assay suitable for screening applications, including the identification and/or characterization of candidate therapeutics for PCT, SCRT, ERT, GT and lysosomal enhancement. Methods Cell Lines Primary fibroblasts derived from MPS III patients were either purchased from the Coriell Institute for Medical Research or obtained from the Telethon Genetic Biobank Network, Italy. Control fibroblast cell lines 21836025 from healthy donors were purchased from the Coriell Institute for Medical Research. Reagents All chemicals were of American Chemical Society reagent grade and, unless otherwise stated, from Sigma Aldrich. Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside was purchased from Moscerdam Substrates. Medium for cell culture consisted of DMEM High Glucose, supplemented with 20% fetal bovine serum, 2 mM L-Glutamin and 100 U/ ml penicillin/100 mg/ml streptomycin. N-acetylglucosaminidase Activity Assay Sample fibroblasts in culture plates were trypsinized, counted with a Neubauer hemocytometer and diluted in their standard medium to the required cell concentration. Then, 100 ml of this suspension were plated in each well of a 96-well plate and incubated at 37uC and 5% 18039391 CO2 overnight to achieve cell attachment. Clear bottom plates were used. Four wells were plated for each condition to be tested. In order to prevent edge effects, columns 1 and 12 were filled with 100 ml PBS. Rows A and H served as background noise control to determine the extent of unspecific fluorescence and were used to incubate cells with 50 ml buffer without substrate. The medium was replaced the next day with fresh medium and plates were incubated at 37uC and 5% CO2. After treatment the medium was removed and cells washed three times with PBS. The assay reaction was started by adding 50 ml of substrate solution to each well. Plates were sealed with plastic wrap to prevent evaporation, covered in aluminum foil in order t
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