| Solution Information | help | |
| Enzyme: | Pantothenate synthetase | |
| inhibitor: | BDBM41551 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Pantothenate Synthethase Protocol for 1 mL cuvet assay Purified recombinant pantothenate synthetase enzyme was provided by Dr. David Eisenberg and Dr. Celia W. Goulding at Howard Hughes Medical Institute and UCLA. 590 uL of PS reagent mix which included NADH, D-pantoate, alpha-alanine, ATP, phosphoenolpyruvate, MgCl2, myokinase, pyruvate kinase, and lactate dehydrogenase in buffer was added to each UV-VIS semimicro cuvet. Compounds were then added to the cuvets in 10 uL volumes. The reaction was initiated with the addition of 400 uL of PS, diluted in assay buffer. The final concentrations in the reaction were 0.5 mM NADH, 5 mM pantoic acid, 10 mM MgCl2, 5 mM alpha-alanine, 10 mM ATP, 1 mM potassium phosphoenolpyruvate, and 18 units/mL each of chicken muscle myokinase, rabbit muscle pyruvate kinase, and rabbit muscle lactate dehydrogenase diluted in 100 mM HEPES buffer (pH 7.8), 1% DMSO, and 10 ug/mL PS in a final volume of 1000 uL. The cuvets was immediately transferred to a CARY 3 | |
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