G6378

Frozen soleus muscle samples (5 mg) were homogenized (1/50) in 2 N NaOH for 2 hr at 37°C and 1 hr at 4°C and 0.2 volumes 7.5 M HCl added. Samples (50 µl) were subjected to glycogen hydrolysis by amyloglucosidase (10 mg/ml) (A7420, Sigma‐Aldrich) in acetate buffer (0.3 M) for 2 hr at 37°C. Released glucose was quantified by the spectrophotometric measurement of NADH production (λ = 340 nm) in the presence of hexokinase (H4502 Sigma) and glucose‐6‐phosphate dehydrogenase (G6378 Sigma), according to the method of Bergmeyer (Bergmeyer & Grassl, 1983 ; Passonneau & Lauderdale, 1974 (link)) and as previously described in (Banzet et al., 2009 (link)). Glycosyl units were quantified by comparison to a standard curve of known glycogen concentration.

All assays (40 μl volume) were carried out at 37 °C. For the measurement of FraB deglycase activity, we used a glucose-6-phosphate dehydrogenase (G6PD)-based coupled assay. The FraB reaction mixture contained 1 mM 6-P-F-Asp, 25 mM HEPES (pH 7.5), 5 mM MgCl₂, 0.1 mM EGTA, 0.5 mM NADP⁺, 0.15 U G6PD (Sigma, G6378). The reaction was initiated by the addition of a defined amount of crude lysates (up to 40% of the assay volume) obtained from wild-type or mutant Salmonella strains. The NADPH generated by G6PD was followed by measuring absorbance at 340 nm and taken as a direct readout of the glucose-6-phosphate produced by FraB. To determine FraD kinase activity, a G6PDH + FraB-based coupled assay was performed. The reaction mixture for the kinase assay contained 1 mM fructose-aspartate (F-Asp), 25 mM HEPES (pH 7.5), 25 mM KCl, 1 mM MgCl₂, 1 mM dithiothreitol, 1 mM ATP, 0.1 mM EGTA, 0.5 mM NADP⁺, 0.3 μM recombinant FraB (Sengupta and Gopalan, unpublished) and 0.15 U G6PD (Sigma, G6378). For both FraB and FraD assays, the reactions were terminated by addition of 6 mM EDTA (final concentration). One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 μmol of NADPH per min. Mean and standard deviation values were calculated from independent assays that used crude lysates from three separate cultures.

KMO activity was measured in brain regions homogenates. 100 µl of homogenized tissues were diluted in 400 µl of KMO buffer activity (containing 100 mM TRIS, 10 mM KCl and 1 mM EDTA, Sigma-Aldrich, USA). Then, 80 µl of homogenate were incubated with 100 µl of assay cocktail (1 mM of NADPH, 3 mM of G6P, 1 U/ml of G6PDH and 100 µM of L-kynurenine sulfate (cat. No. N1630, G6378 and K2750 Sigma-Aldrich respectively)) and incubated at 37 °C for 1 h. The reaction was stopped with 25 µl of perchloric acid 6%. Samples were centrifuged 10 min at 14000 g. 40 µl of supernatants were injected to HPLC and 3HK levels were detected with the above mentioned method^{62 (link)}. The results were presented as pmoles of 3-HK/h/mg of protein.