Coomassie dye binding assay

10 mg of mouse brain cortex was homogenized in buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol, and 0.2% Triton X-100 and centrifuged at 14,000 rpms for 1 min at 4°C. Following centrifugation, the supernatant was removed and used for Naglu, β-gluc, and β-Hexa enzyme assays using the 4-MU fluorometric assay and normalized to total protein as previously described(Benitez and Sands, 2017 (link)). In brief, 1-5 uL of supernatant was added to 100 μl of 5 mM 4-Methylumbelliferyl-β-D-glucuronide hydrate (β-Gluc substrate) (Sigma-Aldrich, Cat# M9130), 4-Methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide (HexA substrate) (Sigma-Aldrich, Cat# 454428) or 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (Naglu Substrate) (Sigma-Aldrich, Cat# M2133) respectively, and incubated at 37°C for 1 h. The reactions were stopped by adding 1 ml of 0.1 M Na₂CO₃. Substrate cleavage was measured at 448 nm emission and 365 nm excitation in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi) using a standard curve ranging from 0.5 to 5 mM of 4-methylumbelliferone (Sigma-Aldrich, Cat# M1381). The values were normalized to total protein measured using a Coomassie dye-binding assay (Bio-Rad Laboratories).

Samples of the same organs used for staining (whole kidney, one brain hemisphere, liver, spleen, and heart) were flash frozen in liquid nitrogen and homogenized in buffer as previously described [13 (link)]. Following centrifugation the supernatant was incubated with the fluorogenic substrate 4-methylumbelliferyl-6-thiopalmitoyl-β-glucoside in a 37°C water bath for 1 h. The reaction was stopped with 500 μL of a 0.1 M sodium carbonate/sodium bicarbonate buffer. Fluorescence emission was measured at 448 nm following excitation at 365 nm in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi, Pleasanton, CA). Measurements were compared to a standard curve ranging from 0.5 to 5 mM of 4-methylumbelliferone (4-MU). PPT1 activity was normalized to total protein measured using a Coomassie dye-binding assay (Bio-Rad Laboratories, Hercules, CA) and data are expressed as nmol of substrate cleaved per mg of protein per h (nmol/mg/h).