Brain samples were homogenized in 250mM sucrose, 10mM Tris (pH 7.4), 1mM ethylenediaminetetraacetic acid supplemented with protease inhibitors (1mM phenylmethanesulfonyl fluoride, 1μg/ml pepstatin A, 1μg/ml leupeptin), and 1mM NaVO4. Homogenate was diluted to 2mg/ml in water and sonicated, and GCase activity was determined in samples (20μg protein) by hydrolysis of 5mM 4-methylumbelliferyl-β-D-glucopyranoside in McIIvaine buffer (pH 5.4) in the presence of 22mM sodium taurocholate at 37°C for 1 hour.20 (link) The reaction was stopped by addition of 0.25M glycine (pH 10.4) and 4-methylumbelliferone fluorescence measured at excitation 365nm, emission 450nm.
Measurement of nonlysosomal GCase (GBA2) was performed as above but in the absence of sodium taurocholate and with the addition of 1μM deoxynojirimycin, a GBA2 inhibitor.21 (link)
β-Hexosaminidase was assayed in the above homogenates (2μg protein) using the fluorogenic substrate 4-methylumbelliferyl-2-acetoamido-2-deoxy-6-sulfo-β-D-glucopyransoside (2mM) in sodium citrate buffer (pH 4.2) at 37°C for 30 minutes. The reaction was stopped by addition of 0.25M glycine (pH 10.4), and fluorescence was measured as above.22 (link)
Measurement of nonlysosomal GCase (GBA2) was performed as above but in the absence of sodium taurocholate and with the addition of 1μM deoxynojirimycin, a GBA2 inhibitor.21 (link)
β-Hexosaminidase was assayed in the above homogenates (2μg protein) using the fluorogenic substrate 4-methylumbelliferyl-2-acetoamido-2-deoxy-6-sulfo-β-D-glucopyransoside (2mM) in sodium citrate buffer (pH 4.2) at 37°C for 30 minutes. The reaction was stopped by addition of 0.25M glycine (pH 10.4), and fluorescence was measured as above.22 (link)
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