Aspergillus niger amylo α 1 4 α 1 6 glucosidase

For GAA activity assays, the CNS, heart, liver, and skeletal muscles were rapidly dissected after euthanasia and PBS perfusion. Brains were sectioned into four coronal slabs of ~2 mm thickness and the spinal cord into two coronal slabs. All tissues were then snap-frozen in liquid nitrogen, and stored at −80 °C until biochemical analyses were performed. Tissues were homogenized in a phosphate buffer, homogenates were centrifuged at 13,000 rpm for 10 min at 4 °C and the resulting supernatant was assayed for GAA activity by measuring cleavage of 4-methylumbelliferyl-α-D-glucopyranoside aſter incubation for 1 h at 37 °C as previously described [46 (link)]. Protein concentration was measured using Bicinchoninic Acid method per manufacturer’s instructions (B9643, Sigma-Aldrich). Biochemical measurement of glycogen content was then performed as described elsewhere [20 (link)]. Tissue extracts were boiled for 3 min and incubated at 54 °C for 1 h in the presence or absence of Aspergillus niger amylo-α-1,4-α-1,6 glucosidase (5 U/ml; Roche, Mannheim, Germany) which converts glycogen to glucose. Samples were centrifuged and glucose level was determined in the supernatant using Glucose RTU kit (Biomerieux, Lyon, France) per manufacturer’s instructions.

For the biochemical analysis, the TA and TB muscles were rapidly dissected, frozen in liquid nitrogen and stored at − 80 °C until processing. The tissues were homogenized in phosphate buffer containing protease inhibitors (Roche, Mannheim, Germany) using Precellys® (Ozyme, Montigny Le Bretonneux, France). The homogenates were centrifuged at 13,000 rpm for 10 min at 4 °C, and the resulting supernatant was used for the biochemical measurement of the glycogen content as described elsewhere [28 (link)]. Briefly, the tissue extracts were boiled for 3 min and incubated at 54 °C for 1 h with or without 5 U/mL Aspergillus niger amylo-α-1,4-α-1,6 glucosidase (Roche, Mannheim, Germany), which converts glycogen to glucose. The samples were centrifuged, and the glucose level in the supernatant was determined using an AmplexRed® Glucose Assay Kit (A22189, Invitrogen, Cergy-Pontoise, France) per the manufacturer’s instructions.