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. 2009 Jun 9;284(32):21526–21535. doi: 10.1074/jbc.M109.014761

FIGURE 2.

FIGURE 2.

Expression, enzymatic activities of recombinant Arabidopsis GalA-kinase (GalAK), Gal-kinase (GalK), and identification of reaction products. A, SDS-PAGE of total soluble protein isolated from E. coli cells expressing Arabidopsis recombinant GalAK (lane 2), control empty vector (lane 6), or Arabidopsis GalK (lane 4), and of nickel-column purified recombinant GalAK (lane 3) and GalK (lane 5). B, HPLC chromatogram of GalAK coupled-based assay. A distinct UDP-GalA peak (marked by arrow #1, in panel 1) is detected in GalAK reaction but not in control (empty vector control cells, panel 2). The peak eluted at 15.2 min (arrow #1, in panel 1) was collected and confirmed after analysis by 1H NMR (panel 4) as UDP-α-GalA. For detailed proton NMR spectra of GalAK enzymatic product, see supplemental Fig. S1, A and B. The HPLC peaks in B (panels 1–3) are ATP (18 min), UTP+ADP (16.3 min), and UDP-GalA (15.2 min). The minor peak at 14.8 min is AMP contamination. C, HPLC chromatogram of GalK coupling-based assay. A UDP-Gal peak (marked by arrow #2 in panel 5) is detected in reaction contained GalK but not in empty vector control cells (panel 6). The peak eluted at 12.2 min (arrow #2, in panel 5) was collected and confirmed after analysis by 1H NMR (panel 8) as UDP-α-Gal. For detailed proton NMR spectra of the enzymatic product, see supplemental Fig. S2, A and B. The HPLC peaks in C (panels 5–7) are ATP (18 min), UTP+ADP (16.3 min), and UDP-Gal (12.2 min). The minor peak at 14.8 min is AMP contamination.