Ldh enzyme

PYK was measured by coupled assay with lactate dehydrogenase (LDH). The assay buffer contained 50 ​mM Tris-HCl, pH 7.5 (at 55 ​°C), 5 ​mM dithiothreitol (DTT), 10 ​mM KCl, 12 ​mM MgCl₂, 10 ​mM ADP, 0.1 ​mM 3-phosphoglyceric acid (3PG), 5 ​mM PEP, 12 U LDH enzyme (Sigma L2500) and 0.3 ​mM NADH. Water was added to a final volume of 1.0 ​mL. PEP was converted to pyruvate, which was converted to lactate by LDH with the concomitant reduction of NADH to NAD⁺. The decrease in NADH concentration over time was measured at 340 ​nm by spectrophotometer. The reaction could be started with either ADP or PEP. For routine assays it was started with PEP. The assay is sensitive to MgCl₂ levels and did not work when MgCl₂ was lower than 2 ​mM.

PPDK was measured by coupled assay with LDH. The assay buffer contained 50 ​mM Tris-HCl, pH 7.5 (at 55 ​°C), 5 ​mM DTT, 0.3 ​mM NADH, 5 ​mM MgCl₂, 20 ​mM NH₄Cl, 2 ​mM PEP, 12 U LDH enzyme (Sigma L2500), 2 ​mM AMP and 1 ​mM pyrophosphate (PPi). The reaction was started with the addition of PP_i, although it could also be started by adding AMP or PEP. The decrease in NADH concentration over time was measured at 340 ​nm by spectrophotometer. NH₄Cl is an activator for PPDK activity and is essential to the enzyme assay. At 55 ​°C, the mixture of 5 ​mM MgCl₂ and 1 ​mM PP_i forms a precipitate after 1 ​h, so the assay must be done within 1 ​h for accurate measurement. Slight increases in the concentration of either compound resulted in rapid precipitation.