Article Figures & Data
Figures
- FIG. 1.
Effects of divalent cations on UgpQ activity. The glycerophosphodiester phosphodiesterase activity was measured in the presence of 1 mM GPC and various concentrations of divalent cations. The divalent cations shown are MnCl2 (□), CoCl2 (▪), MgCl2 (○), and CaCl2 (•). The data are the means of triplicate measurements. The error bars represent standard deviations.
- FIG. 2.
Effect of pH on UgpQ activity. The reaction mixture contained 50 mM buffer at a pH range from 5.5 to 10, 1 mM GPC, 5 mM MgCl2, and 2 μg/ml UgpQ. The reaction rate was maximal at pH 7.5. The pH dependency of the enzyme reaction showed a typical bell-shaped profile. The buffers used for the experiments were MES-NaOH (▪), PIPES-NaOH (▴), MOPS-NaOH (▾), HEPES-NaOH (○), TAPS-NaOH (•), and CHES-NaOH (□). The data are the means of triplicate experiments. The error bars represent standard deviations.
- FIG. 3.
Induction of glycerophosphodiester phosphodiesterase activity in phosphate-starved E. coli. (a) Growth curve of wild-type E. coli cultured in phosphate-rich and -poor media. E. coli was cultured in a synthetic medium, as described in Materials and Methods. Briefly, starvation for phosphate was provoked by cultivation in a medium containing 0.16 mM Pi (○). The phosphate-rich medium contained 0.6 mM Pi (•). The data are representative of three independent experiments that produced similar results. OD600, optical density at 600 nm. (b and c) Glycerophosphodiester phosphodiesterase activities in the soluble and membrane fractions of wild-type (WT) and UgpQ-deficient E. coli. The soluble (b) and membrane (c) fractions of wild-type and UgpQ-deficient E. coli cells that were cultured for 9 h were assayed for glycerophosphodiester phosphodiesterase activity at 35°C in the presence of 1 mM EDTA, 5 mM MgCl2, 5 mM CaCl2, or 100 μM MnCl2. The data are the means of triplicate experiments. The error bars represent standard deviations.
- FIG. 4.
SDS-PAGE and immunoblot analysis. (a) SDS-PAGE of purified recombinant UgpQ. A single band was observed at 27 kDa by Coomassie brilliant blue staining. (b) Detection of UgpQ in phosphate-starved E. coli by immunoblotting. The soluble fractions of wild-type (WT) and UgpQ-deficient E. coli under phosphate-starved (0.16 mM) and -rich (0.6 mM) conditions (4 μg of protein in each lane) were subjected to immunoblot analysis using rabbit antiserum against UgpQ (1:2,000 dilution).
- FIG. 5.
Proposed metabolic pathway of glycerophosphodiesters by UgpQ and GlpQ in E. coli. GPD, glycerophosphodiesters; IM, inner membrane; OM, outer membrane; R-OH, ethanolamine, glycerol, choline, serine, inositol, etc.
