A heptameric peptide purified from Spirulina sp. gastrointestinal hydrolysate inhibits angiotensin I-converting enzyme- and angiotensin II-induced vascular dysfunction in human endothelial cells

Figures

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  Figure 1. (A) Fast protein liquid chromatography (FPLC) of tryptic hydrolysate loaded (2 ml) onto a HiPrep™ 16/10 DEAE FF anion-exchange column. (B) ACE inhibitory activity of each fraction from FPLC.

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  Figure 2. (A) Reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of the potent angiotensin I (Ang I)-converting enzyme (ACE) inhibitory activity fraction isolated from fast protein liquid chromatography (FPLC). (B) ACE inhibitory activity of each fraction from RP-HPLC.

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  Figure 3. Identification of the molecular mass and amino acid sequence of the angiotensin I (Ang I)-converting enzyme (ACE) inhibition peptide using quadrupole time-of-flight mass spectroscopy with an electrospray ionization (ESI) source.

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  Figure 4. Lineweaver-Burk plots for determination of inhibitory mode of purified peptide on angiotensin I (Ang I)-converting enzyme (ACE). ACE inhibitory activity was determined in the presence or absence of purified peptide as described in the text using N-Hippuryl-His-Leu tetrahydrate (HHL) as the enzyme substrate.

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  Figure 5. The binding site of the purified peptide in the cleft of ACE. ACE is represented by ribbons, and zinc atom in active site is represented by a gray sphere. Ang II and the purified peptide are represented by thick sticks, and amino acids of ACE involved in hydrogen bonds are represented by thin sticks. (A) Carbon, nitrogen, oxygen, sulfur and hydrogen atoms are colored by cyan, blue, red, yellow and white, respectively. Carbon atoms of the purified peptide are colored by green. N and C indicate the N- and C-terminus of the enzyme, respectively. The snapshot is taken at 121.6 nsec. (B) Mixed non-competitive inhibition mechanism of ACE by the purified peptide. The broken lines indicate hydrogen bonds (I, inhibitor, or the purified peptide; ACE, angiotensin I-converting enzyme; Ang I, angiotensin I, or substrate; Ang II, angiotensin II, or product; ESI, enzyme-substrate-inhibitor complex; EPI, enzyme-product-inhibitor complex, dead-end complex). (C) Ratio of the presence of hydrogen bond pairs during the simulation time.

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  Figure 6. (A) Effect of peptide on cell viability and (B) nitric oxide (NO) production in angiotensin II (Ang II)-stimulated Ea.hy926 cells. The cells were pretreated for 1 h with various concentrations (62.5, 125 and 250 µM) of the purified peptide. Ang II (1 µM) was then added, and the cells were incubated for 24 h. The cytotoxicity was determined by MTT assay. *p<0.05 indiciate significant differences compared with the Ang II-stimulated group.

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  Figure 7. Inhibitory effects of peptide on angiotensin II (Ang II)-induced reactive oxygen species (ROS) generation in EA.hy926 cells. Cells were pre-treated with various concentrations of the purified peptide (62.5, 125 and 250 µM) for 30 min, and then treated with Ang II for 30 min. The intracellular ROS generated was detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. The values are expressed as the means ± standard deviation of triplicate experiments. *p<0.05 indiciate significant differences compared with the Ang II-stimulated group.

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  Figure 8. Inhibitory effect of the purified peptide on angiotensin II (Ang II)-induced inducible nitric oxide synthase (iNOS) and endothelin-1 (ET-1) in EA.hy926 endothelial cells. EA.hy926 cells were stimulated with Ang II for 24 h in the presence of the purified peptide (62.5, 125 and 250 µM). The values are expressed as the means ± standard deviation of triplicate experiments. *p<0.05 indiciate significant differences compared with the Ang II-stimulated group.

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  Figure 9. Inhibitory effect of the purified peptide on the protein level of mitogen activated protein kinases (MAPKs) in EA.hy926 endothelial cells. EA.hy926 cells were stimulated with angiotensin II (Ang II) for 30 min in the presence of the purified peptide (62.5, 125 and 250 µM). The expression levels of MAPKs were determined by western blot analysis. The values are expressed as the means ± standard deviation of triplicate experiments. *p<0.05 indiciate significant differences compared with the Ang II-stimulated group.

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  Figure 10. Summary of experimental results from the present study in the inhibition of angiotensin I (Ang I)-converting enzyme (ACE) and vascular dysfunction.