Effect of VPA on the secondary structure of rhPE.

<p>(<b>A</b>) Three VPA concentrations (6, 12 and 24 mM) were titrated into 2.5 µM rhPE. VPA caused a significant change in the secondary structure of the enzyme. (<b>B</b>) A high concentration of ZPP (1 µM) was added to the 2.5 µM rhPE/24 mM VPA mixture. ZPP did not cause any change in the secondary structure of the enzyme. (<b>C</b>) Adding 1 µM ZPP to 2.5 µM rhPE does not cause any secondary protein structure change. Adding 24 mM VPA on top of that mixture doesn't cause any structure change.</p

Effect of VPA on the activity of rhPE.

<p>(<b>A</b>) Activity of 10 nM purified rhPE was measured in presence of ten doses of VPA or lithium ranging from 0.2–10 mM. Relative activity of rhPE in the presence of VPA or lithium is shown as a percentage of activity in the absence of VPA and lithium. VPA showed a <i>K_i</i> of approximately 1 mM. lithium showed no effect on PE activity. (<b>B</b>) Inhibition curves of three VPA concentrations (0.8, 1.6 and 3.5 mM) were obtained by incubating VPA with 10 nM rhPE during 90 min at 37 °0. (<b>C</b>) A Lineweaver-Burk plot was made based on the rhPE activity assays with 0.8 and 1.6 mM VPA as inhibitor. Enzyme activity was measured with increasing substrate (Suc-Gly-Pro-pNA) concentrations, ranging from 0.2–10 mM. The velocity was calculated as mM * min⁻¹ * ml⁻¹. Data are shown as the mean ± S.E.M. (n = 3 per group).</p

VPA decreases cigarette smoke-induced neutrophil influx in BAL fluid of mice.

<p>(<b>A</b>) Total cell numbers, (<b>B</b>) macrophages and (<b>C</b>) neutrophils in the BAL fluid of mice exposed to air or whole body cigarette smoke twice daily during 5 days. The mice received vehicle (PBS) or VPA (100 µg/70 µl PBS) by oropharyngeal aspiration 15 minutes prior to air/smoke exposure. (<b>D</b>) acPGP was measure in the BAL fluid. (<b>E</b>) PE activity was measured in the BAL fluid and compared to control (PBS treated/air exposed mice). N = 5–10 animals per group. Values are expressed as mean+/−S.E.M. **P≤0.01, ***P≤0.001, ns p>0.05.</p

NMR spectra of the carbobenzoxy-group of ZPP.

<p>(<b>A</b>) 10 µM ZPP was added to 10 µM rhPE, 10 mM VPA was added to that mixture. A shift of the free ZPP peaks at 7.28 and 7.20 ppm to the left is seen (indicated in the red and green bars respectively). (<b>B</b>) 10 µM ZPP was added to 10 µM rhPE. A shift of the free ZPP peaks at 7.28 and 7.20 ppm to the left is seen. (<b>C</b>) 10 µM ZPP was measured without enzyme. The peaks at 7.28 and 7.20 ppm are the free ZPP fractions. (<b>D</b>) 10 mM VPA was added to 10 µM rhPE, 10 µM ZPP was added to that mixture. Note that the free ZPP peaks at 7.28 and 7.20 ppm do not show a shift to the left; there are no peaks in the highlighted areas.</p

Inhibition of PGP generation by VPA.

<p>(<b>A</b>) Dialyzed Collagen Type I and II were incubated with the lysate of 4.6 * 10⁶ PMN at 37°C for 20 hours to generate PGP. (<b>B</b>) PE activity was measured 30 minutes after incubation of lysate/collagen/VPA mixture and compared to control (no VPA). Data are shown as the mean ± S.E.M. (n = 5–10 per group). Representative of 4 experiments. # p<0.0001 compared to control, $ p<0.01 compared to control, ** p<0.01, ns p>0.05.</p