Article Figures & Data
Figures
- Fig. 1.
R- and S-enantiomers of 19-HETE. Both enantiomers represent an example of stereoisomerism of P450-mediated arachidonic acid metabolites. Incorporation of an hydroxyl group in the carbon skeleton of arachidonic acid generates a chiral center, hence there are two enantiomers for each HETE metabolite.
- Fig. 2.
Inhibitory effect of 19(R)-HETE and 19(S)-HETE on EROD activity mediated by human recombinant CYP1B1. In 96-well solid black polystyrene plates, the reaction mixture containing 100 mM potassium phosphate (pH 7.4) buffer supplemented with 5 mM magnesium chloride hexahydrate and 1 pmol of human CYP1B1 was incubated with 10–100 nM 7-ER. In addition, either 19(R)-HETE (A) or 19(S)-HETE (B) at 0, 5, 10, 20, or 40 nM concentration was added to the reaction. The reaction was initiated by the addition of 100 μl of 2 mM NADPH, the fluorescent signal related to the formation of resorufin was measured every minute (excitation and emission wavelengths of 550 and 585 nm, respectively) for 30 minutes at 37°C using a BioTek Synergy H1 Hybrid. The quantity of formed resorufin was measured by the construction of standard curve of 0–200 nM resorufin dissolved in the same incubation buffer. Each point represents the mean of six independent experiments ± S.E.
- Fig. 3.
Dixon plots representing the inhibitory effect of 19(R)-HETE and 19(S)-HETE on EROD activity mediated by human recombinant CYP1B1. The y-axis represents the reciprocal of EROD activity expressed in picomoles of resorufin/picomoles CYP1B1/minute and the x-axis represents 19(R)-HETE (A) or 19(S)-HETE (B) concentrations in nanomolar concentrations in the presence of increasing concentration of the substrate 7-ER (10–100 nM). Each point represents the mean of six independent experiments ± S.E.
Tables
- TABLE 1
The mean values and S.E. of kinetic parameters of resorufin formation by human recombinant CYP1B1 in the absence and presence of 19(R)-HETE or 19(S)-HETE
Results are presented as mean and S.E., of at least three individual experiments. Km, Vmax and Ki mean ± S.E. were determined by the Enzyme Kinetics module of GraphPad Prism, version 5.01.
Km Vmax Ki 19(R)-HETE 19(S)-HETE nM pmol/pmol P450 per minute nM nM 86.6 ± 10.4 16.7 ± 1.1 89.1 ± 11.5 37.3 ± 5.5 - TABLE 2
The mean values and S.E. of 19(R)-HETE or 19(S)-HETE levels calculated as a percentage of the metabolite level at the beginning of the experiment (T0)
Data are represented as mean ± S.E. where n = 3.
19(R/S)-HETE levels as a percentage of their levels at zero time (%) Time interval (Tminute) T0 T10 T20 T30 19(R)-HETE 100 ± 17.1 98.6 ± 7.8 96.3 ± 1.3 98.5 ± 8.9 19(S)-HETE 100 ± 5.9 99.7 ± 1.3 100.1 ± 0.9 98.9 ± 4.6
Data Supplement
- Supplemental Figures -
Supplemental Figure 1 - Representative LC-MS chromatograms showing the peaks of 19(R)-HETE and internal standard at the start of the experiment (a), after 10 min (b), 20 min (c) and 30 min (d)
Supplemental Figure 2 - Representative LC-MS chromatograms showing the peaks of 19(S)-HETE and internal standard at the start of the experiment (a), after 10 min (b), 20 min (c) and 30 min (d).
- Supplemental Figures -
