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PGK1 enzyme activity measurements

XK Xin-Le Kang
YL Yan-Xue Li
DD Du-Juan Dong
JW Jin-Xing Wang
XZ Xiao-Fan Zhao
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PGK1 WT and PGK1 variants were overexpressed in HaEpi cells individually and PGK1-His were pulled down for the PGK1-catalyzed reaction to investigate the directionality of the PGK1 reaction. PGK1 activity was measured at room temperature in kinetic mode for 5 min by coupling with GAPDH. The absorbance at 340 nm was measured based on changes in NADH concentrations with a BioTek Synergy Neo Multi-Mode Plate Reader (BioTek, USA). PGK1 was isolated from H. armigera cell lysates or fat body and subjected to a PGK1-catalyzed reaction of 3-PG production assays in a reaction buffer containing 5 mM KH2PO4 (pH 7.0), 1 mM glyceraldehyde 3-phosphate (3-GAP), 0.3 mM β-nicotinamide adenine dinucleotide (β-NAD), 0.2 mM ADP, 5 mM MgSO4, 100 mM glycine, and 5 ng/mL GAPDH.

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