The PFK activity assay was performed in accordance with manufacturer’s instructions (MAK093; Sigma-Aldrich) with purified V. cholerae PFK. This assay quantifies PFK activity by measuring its capacity to convert fructose-6-phosphate to fructose 1,6-diphosphate. This reaction produces ADP, which is converted to AMP and NADH by the reaction mix. The resulting NADH reduces a probe resulting in a colorimetric (450 nm) product proportional to the PFK activity of the sample. Specific activity was calculated in pmol NADH/mg protein. Racemase activity assays using purified BsrV protein were performed essentially as described in ref. 39 (link) (see Extended Data Fig. 7 for schematic representation of both activities). To assess the effect of nitrite under low pH on PFK and BsrV activities, 5 μg of each enzyme were incubated for 4 hours in 50mM Tris HCl at pH 6 in the presence or absence of 20 mM sodium nitrite, and an aliquot of 0.1 μg protein collected and added into each specific activity reaction mix.
Mak093
Manufactured by Merck Group
MAK093 is a laboratory instrument designed for automated cell analysis. It can perform rapid and accurate cell counting, viability assessment, and size measurement.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using mak093
1
Effect of Nitrite on PFK and Racemase
2
Measurement of Citrate Synthase and PFK
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Citrate synthase and phosphofructokinase (PFK) activity were assayed in muscle lysates using commercially available kits (CS: CS0720, PFK: MAK093, St. Louis, MO, Sigma-Aldrich) according to the manufacturer’s instructions as described previously (42). All activities were normalized to protein concentration as described above and expressed in international mU·mg−1 protein.
3
Effect of Nitrite on PFK and Racemase
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The PFK activity assay was performed in accordance with manufacturer’s instructions (MAK093; Sigma-Aldrich) with purified V. cholerae PFK. This assay quantifies PFK activity by measuring its capacity to convert fructose-6-phosphate to fructose 1,6-diphosphate. This reaction produces ADP, which is converted to AMP and NADH by the reaction mix. The resulting NADH reduces a probe resulting in a colorimetric (450 nm) product proportional to the PFK activity of the sample. Specific activity was calculated in pmol NADH/mg protein. Racemase activity assays using purified BsrV protein were performed essentially as described in ref. 39 (link) (see Extended Data Fig. 7 for schematic representation of both activities). To assess the effect of nitrite under low pH on PFK and BsrV activities, 5 μg of each enzyme were incubated for 4 hours in 50mM Tris HCl at pH 6 in the presence or absence of 20 mM sodium nitrite, and an aliquot of 0.1 μg protein collected and added into each specific activity reaction mix.
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