Characterization of recombinant proteins and inhibition of GhPME2 and GhPME31 by GhPMEI3. A, Production of GhPMEI3, GhPME2, and GhPME31 in transgenic E. coli. Lane 1, purified GhPME2; lane 2, purified GhPMEI3; lane 3, purified GhPME31; lane 4, purified GhPMEI3 detected by western-blot analysis with anti-His antibodies. B, MALDI-TOF mass spectra of the GhPME2 PRO region. C, MALDI-TOF mass spectra of the GhPME31 PME catalytic domain. The obtained recombinant protein spots were digested by trypsin, and the resulting peptides were analyzed as described in “Materials and Methods.” D, Inhibition of GhPME2 and GhPME31 activity by GhPMEI3. The experiments were carried out using 1 mU of GhPME2 or GhPME31, with different amounts of purified GhPMEI3. The same reaction was performed with heat-denatured (95°C, 5 min) PME; water instead of PME was used as a negative control. The results are presented as the means ± se (n = 3). Asterisks indicate significant differences compared with reactions lacking GhPMEI3 (least significance difference, lsd, test; *P < 0.05, **P < 0.01). E, Inhibition of GhPME2 and GhPME31 by GhPMEI3; 2 μg of GhPMEI3 was added to the reaction mixture 10 min after the initiation of the reaction. The same reaction was performed with heat-denatured (95°C, 5 min) PME. Water instead of PME was used as a negative control. The results indicate the amounts of the reaction products (NADH). Means marked with the same letter were not significantly different, according to Tukey’s HSD test at P < 0.05.