FIGURE 1.

Purification and identification of the protein with zeatin cis–trans isomerase activity. (A) Typical conversion of zeatin isomers (0.2 mM) by maize kernel extracts after incubated in 100 mM McIlvaine buffer containing 20 mM MgCl₂, 0.1 mM FAD and 2.0 mM dithiothreitol, at 37°C under white fluorescent light for 2 h; determined by UFLC; graph depicts average of three technical replicates with standard deviation. (B) SDS-PAGE of the individual protein purification steps: 1, crude extract; 2, extract after protein precipitation; 3, fraction from DEAE-Sepharose; 4, High Q; 5, Concanavalin A; 6 Resource Q; 7 Bio-Scale CHT5-I; 8 HiTrap Blue HP; 9, MW marker, 100 and 50 kDa indicated by arrows. (C) Isoelectric focusing gel with the sample after HiTrap Blue HP (silver-stained lane); the rest of the gel slab was sliced, proteins extracted and activity detected in the band indicated by the asterisk. (D) SDS-PAGE of the isolated active protein: 1, MW marker, 100 and 50 kDa indicated by arrows; 2, purified active protein; the vertical black line denotes combining of two image parts. (E) SDS-PAGE of the isolated recombinant ZmNPP protein: 1, MW marker; 2, purified enzyme from the yeast growth medium.