Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the chromosome 1 gene cluster coding for the four mouse AOX enzymes, showing the relative length and position of the four genes encoding the indicated AOX enzymes. The four genes are transcribed in the same direction, as indicated. The numbers below each gene indicate the number of amino acids for each encoded protein.
- Fig. 2.
Characterization of the mAOX1, mAOX3, mAOX4, and mAOX2 proteins expressed in E. coli. The main graphs illustrate the size-exclusion chromatograms (absorbance at 450 nm) of the indicated proteins isolated from E. coli. The UV/Vis spectra of purified (A) mAOX1, (B) mAOX3, (C) mAOX4, and (D) mAOX2 are shown in the right-most inset of each panel. These insets show the UV/Vis spectrum of air-oxidized enzymes. The UV/Vis spectra were recorded in 50 mm Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0). The 10% SDS–polyacrylamide gels of the indicated proteins after staining with Coomassie Blue are shown in the left-most inset of each panel.
- Fig. 3.
Molybdenum and iron saturation of purified mAOX proteins. After purification, the molybdenum and iron content of the indicated enzymes were determined by inductively coupled plasma optical emission spectroscopy. The iron content corresponds to saturation with both, FeSI and FeSII clusters. The percentage values are calculated on the basis of the theoretical values of Moco and 2×[2Fe–2S] clusters saturation.
- Fig. 4.
Activity staining of mAOX enzymes. The figure illustrates 7% native polyacrylamide gels of purified mAOX1, mAOX3, mAOX4, and mAOX2 (10 μg) run under nondenaturing conditions: Gels were stained with (A) Coomassie Brilliant Blue or 1 mM Nitro blue tetrazolium chloride following incubation with (B) 500 μM phthalazine for 5 minutes and (C) 500 μM pyridoxal-5-monophosphate overnight.
- Fig. 5.
UV/Vis spectra of mAOX enzymes reduced with NADH under anaerobic conditions. The figure illustrates UV/Vis spectra of the indicated mAOX enzymes following purification and challenge with 10 μM oxidized (solid line) and reduced with 0.5 mM NADH after 1 minute (dashed line) and 20 minute (dashed dotted line). The spectra were recorded in 50 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA (pH 8.0). (A) mAOX1, (B) mAOX3, (C) mAOX4, and (D) mAOX2.
Tables
- TABLE 1
General characterization of mAOX1, mAOX3, mAOX4, and mAOX2 after expression in E. coli TP1000 cells
Sample Specific Activitya Yield Ratio (280/450) Ratio (450/550) As Purified After Chemical Sulfuration nmol min−1 mg−1 nmol min−1 mg−1 mg/l of E. coli Culture mAOX1 N.D. 146.7 ± 19.5 1.2 ± 0.2 5.7 3.2 mAOX3b 104b 158b 0.8b 5.2b 3.1b mAOX4 48.4 ± 8.1 585.2 ± 37.3 1.1 ± 0.4 5.4 3.1 mAOX2 N.D. 880.4 ± 27.7 0.8 ± 0.2 5.0 3.2 - TABLE 2
Steady state kinetic parameters of mAOX1, mAOX3, mAOX4, and mAOX2
Steady-state kinetics were corrected to molybdenum saturation of 100%. Kinetic parameters were recorded in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) in the presence of 100 μM DCPIP as electron acceptor. Data are mean values from three independent measurements (±S.D.).
Substrate Kinetic Parameters mAOX1 mAOX3 mAOX4 mAOX2 Pyridoxal kcat (min−1) N.D. N.D. N.D. N.D. KM (μM) kcat / KM (min−1 × μM−1) Vanillin kcat (min−1) 449.9 ± 26.4 159.5 ± 3.8 15.9 ± 2.2 80.4 ± 11.5 KM (μM) 104.0 ± 17.7 4.6 ± 0.5 5.4 ± 0.5 2.3 ± 0.7 kcat / KM (min–1. μM−1) 4.3 34.5 2.9 42.1 p-DMAC kcat (min–1) 116.2 ± 7.3 88.5 ± 4.8 30.5 ± 4.2 44.8 ± 3.7 KM (μM) 3.3 ± 0.2 0.5 ± 0.1 0.8 ± 0.1 0.4 ± 0.1 kcat / KM (min–1. μM−1) 34.1 196.7 36.3 125.3 N1-Methylnicotinamide kcat (min−1) N.D. 19.6 ± 4.8 N.D. 49.1 ± 5.7 KM (μM) 27.9 ± 2.5 9.4 ± 3.2 kcat / KM (min–1. μM−1) 0.7 5.2 Salicylaldehyde kcat (min–1) 137.5 ± 2.7 213.0 ± 23.4 205.2 ± 6.7 221.8 ± 49.7 KM (μM) 89.6 ± 2.9 3.5 ± 0.1 13.2 ± 2.4 21.1 ± 12.0 kcat / KM (min–1. μM−1) 1.5 60.4 15.6 10.5 Retinaldehyde kcat (min−1) 122.7 ± 5.1 46.5 ± 6.8 10.6 ± 1.2 27.9 ± 0.4 KM (μM) 16.5 ± 2.4 32.1 ± 5.6 12.6 ± 2.3 26.4 ± 1.57 kcat / KM (min–1. μM−1) 7.4 1.4 0.8 1.1 N.D., no activity was detectable
- TABLE 3
Superoxide production of mAOX1, mAOX3, mAOX4, and mAOX2
Superoxide production of enzymes was calculated by following the reduction of 100 μM cytochrome c at 550 nm in the presence of 50 μM substrate (p-DMAC or vanillin) in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) buffer. Substrate consumption was monitored directly by the decrease in UV absorbance at wavelengths of 398 and 347 nm for p-DMAC and vanillin, respectively, in the presence of oxygen as electron acceptor. Extinction coefficients used are 21,000 M–1cm−1 for cytochrome c, 30,500 M–1cm−1 for vanillin, and 25,100 M–1cm−1 for p-DMAC.
Substrate mAOX1 mAOX3 mAOX4 mAOX2 Vanillin Substrate consumption (U μmol−1 of enzyme) 51.85 29.72 9.17 5.78 Superoxide formation (U μmol−1 of enzyme) 16.45 6.92 1.88 2.42 Ratio (%) 31.7 23.3 20.4 41.8 p-DMAC Substrate consumption (U μmol−1 of enzyme) 142.28 27.44 19.37 15.83 Superoxide formation (U μmol−1 of enzyme) 43.47 5.25 3.84 6.21 Ratio (%) 30.6 19.1 19.9 39.3 One unit (U) is defined as the oxidation of 1 μmol substrate per minute under assay conditions.
Data Supplement
- Supplemental Data -
Supplemental Figure 1 - The chemical structures of the substrates and formed products
used in this study
- Supplemental Data -
In this issue
Characterization of Aldehyde Oxidase Enzymes from Mouse
