Influence of chelators on the enzyme activity of glyceryl ether monooxygenase, nitric oxide synthase, phenylalanine hydroxylase, and fatty aldehyde dehydrogenase.
A. Solubilised microsomes displaying glyceryl ether monooxygenase (●) and fatty aldehyde dehydrogenase (▾) activity were incubated with 1,10-phenanthroline for 15 min at 25°C by vigorous shaking and assayed for the respective enzyme activity. Formation of pyrenedecanoic acid was detected by fluorescence detection after HPLC based product separation as described under “Materials and Methods”. 1,10-Phenatroline effect on nitric oxide synthase (∎) was tested in stimulated RAW264.7 cells and conversion of radioactively labelled L-arginine to L-citrulline was measured as indicated in the section “Materials and Methods”. Recombinant phenylalanine hydroxylase (▴) was incubated with 1,10-phenanthroline and the resulting enzyme activity was measured by fluorescence detection of the formed tyrosine after HPLC based separation. B. Glyceryl ether monooxygenase from solubilised rat liver microsomes was incubated for 15 min at 25°C by vigorous shaking with 1,10-phenatroline (●), 1,7-phenatroline (□), or 1,10-phenatroline pre-incubated with 1.1 fold molar excess of ferrous iron (Δ). Activity was tested by HPLC based separation of the pyrene-labelled acid and quantification by fluorescence detection. C. Solubilised microsomes displaying glyceryl ether monooxygenase (●) and fatty aldehyde dehydrogenase (▾) activity were incubated with in various concentrations of EDTA for 15 min at 25°C by vigorous shaking and assayed for the respective enzyme activity. Formation of pyrenedecanoic acid was detected by fluorescence detection after HPLC based product separation as described under “Materials and Methods”. EDTA effect on nitric oxide synthase (∎) was tested in stimulated RAW264.7 cells and conversion of radioactively labelled L-arginine to L-citrulline was measured as indicated in the section “Materials and Methods”. Recombinant phenylalanine hydroxylase (▴) was incubated with EDTA and the resulting enzyme activity was measured by fluorescence detection of the formed tyrosine after HPLC based separation. Data are all normalised to the enzyme activities obtained under standard conditions without chelator addition. Mean ± SD values for at least three independent experiments are shown. Levels of significance obtained by one-way ANOVA followed by Bonferroni’s post hoc test are indicated in the figure.

Influence of divalent metal ions on the enzyme activity of glyceryl ether monooxygenase, nitric oxide synthase, phenylalanine hydroxylase, and fatty aldehyde dehydrogenase.
A. Glyceryl ether monooxygenase (●) from solubilised rat liver microsomes, nitric oxide synthase (∎) from stimulated RAW264.7, recombinant phenylalanine hydroxylase (▴), and fatty aldehyde dehydrogenase (▾) from solubilised rat liver microsomes were incubated with increasing concentrations of ferrous iron. For exact description of the enzyme activity assays see section “Materials and Methods”. The same experimental protocol was followed for Ca²⁺ (B), Mn²⁺ (C), Co²⁺ (D), Ni²⁺ (E), Zn²⁺ (F), and Cu²⁺ (G). Data are normalised to the enzyme activities obtained under standard conditions without metal ion addition. Mean ± SD values for at least three independent experiments are shown. Levels of significance obtained by one-way ANOVA followed by Bonferroni’s post hoc test are indicated in the figure.

Influence of N⁵-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin on the enzyme activity of glyceryl ether monooxygenase.
A. Glyceryl ether monooxygenase activity was measured in the presence of five different concentrations of tetrahydrobiopterin (0 - 200 μm) and increasing amounts of the cofactor analogue N⁵-methyltetrahydrobiopterin (● 0 μm; ∎ 100 μm; ▴ 300 μM; ▾ 1 mm). One of three independent experiments is shown. B. Lineweaver-Burk plot of the data shown in panel A. Each data set was analysed by linear regression and V_max and K_M of tetrahydrobiopterin at each individual concentration of N⁵-methyltetrahydrobiopterin were calculated. C. Glyceryl ether monooxygenase was incubated with increasing concentrations (0 μm, 50 μm, 100 μm, 200 μm, 500 μm, and 1000 μm) of the cofactor analogue 4-aminotetrahydrobiopterin. Formation of pyrenedecanoic acid was detected by fluorescence detection after HPLC based product separation as described under “Materials and Methods” and activity was plotted normalised to the enzyme activity obtained under standard conditions without exceptional cofactor addition. Data are presented as means ± SD for 4 independent experiments.