| Assay Method Information | |
| | PEPCK Assay (OAA to PEP-allosteric) |
| Description: | Allosteric binding site. The activity of PEPCK in the direction from OAA to PEP was assayed with 50 mM HEPES (pH 7.5), 10 mM DTT, 4 mM MgCl2, 0.5 mM GTP, 1 mM ADP, 187.5 μM NADH, 45 units of LDH, 50 μg of PK, 0.2 mM MnCl2, and (∼1−2 μg) of PEPCK. The assay mixture was incubated for 5 min at 25 °C and the reaction started with the addition of OAA to a final concentration of 350 μM. A second reaction without PEPCK was run simultaneously to account for the spontaneous decarboxylation of OAA. Inhibition assays with MPA were conducted under both variable OAA and variable GTP concentrations. When the GTP concentration was varied, MgCl2 concentrations were also varied to keep a constant 4:1 metal:nucleotide ratio. |
| Affinity data for this assay | |
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