| Solution Information | help | |
| Enzyme: | Diphosphomevalonate decarboxylase | |
| inhibitor: | BDBM45764 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Diphosphomevalonate Decarboxylase Protocol for 0.5 mL Fluorescence Cuvet Assay Purified recombinant DPM-DC enzyme was provided by Dr. Thomas Leyh, Albert Einstein College of Medicine of Yeshiva University. 236 uL of DPM-DC reagent mix which included NADH, diphosphomevalonate, ATP, phosphoenolpyruvate, MgCl2, KCl, pyruvate kinase, and lactate dehydrogenase in buffer was added to each 0.5 mL fluorescence cuvet. Compounds were then added to the cuvets in 4 uL volumes in DMSO. The reaction was initiated with the addition of 160 uL of DPM-DC, diluted in assay buffer. The final concentrations in the reaction were 0.02 mM NADH, 0.005 mM diphosphomelalonate, 4 mM MgCl2, 50 mM KCl, 0.4 mM ATP, 2 mM potassium phosphoenolpyruvate, 3 units/mL each of rabbit muscle pyruvate kinase and rabbit muscle lactate dehydrogenase, 1% DMSO, and 30 nM DPM-DC enzyme diluted in 50 mM HEPES buffer (pH 7.8) in a final volume of 400 uL. The cuvets were immediately transferred to an Aminco-Bowman Series 2 Lumine | |
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