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. 2022 May 26;27(11):3437.
doi: 10.3390/molecules27113437.

Biochemical, Kinetic and Biological Properties of Group V Phospholipase A2 from Dromedary

Mona Alonazi  1 , Aida Karray  2 , Raida Jallouli  3 , Abir Ben Bacha  1   4
Affiliations

Biochemical, Kinetic and Biological Properties of Group V Phospholipase A2 from Dromedary

Mona Alonazi et al. Molecules. .

Abstract

Secretory group V phospholipase A2 (PLA2-V) is known to be involved in inflammatory processes in cellular studies, nevertheless, the biochemical and the enzymatic characteristics of this important enzyme have been unclear yet. We reported, as a first step towards understanding the biochemical properties, catalytic characteristics, antimicrobial and cytotoxic effects of this PLA2, the production of PLA2-V from dromedary. The obtained DrPLA2-V has an absolute requirement for Ca2+ and NaTDC for enzymatic activity with an optimum pH of 9 and temperature of 45 °C with phosphatidylethanolamine as a substrate. Kinetic parameters showed that Kcat/Kmapp is 2.6 ± 0.02 mM-1 s-1. The enzyme was found to display potent Gram-positive bactericidal activity (with IC50 values of about 5 µg/mL) and antifungal activity (with IC50 values of about 25 µg/mL)in vitro. However, the purified enzyme did not display a cytotoxic effect against cancer cells.

Keywords: biological activities; characterization; kinetics; phospholipase V.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A). Chromatography on RP-HPLC column of the purified DrPLA2-V from dromedary heart. RP-HPLC on a C18 column pre-equilibrated in solvent A, elution was performed using a gradient from 0% to 100% solvent B at a flow rate of 1 mL/min. Solvent A is composed of water/trifluoroacetic acid TFA (1000:1, v/v) and solvent B contained 100% acetonitrile. The gradient is indicated by the dotted line. The absorbance was measured at 280 nm. AU: Arbitrary Units. (B) 15%-SDS-PAGE of pure DrPLA2-V. Lane 1, molecular mass markers (kDa); lane 2, 10 µg of purified DrPLA2-V. (C) NH2 sequence alignment of DrPLA2-V, Miniopteridae family (Miniopterusnatalensis) (XP_016070213.1) (i), human family (homosapiens) (NP_000920.1) (ii), and Hyaenida family (Hyaena hyaena) (XP_039084994.1) (iii). Identical amino acids are shown in red.
Figure 2
Figure 2
Evaluation of pH and temperature effect on activity (A,B) and stability (C,D) of DrPLA2-V.
Figure 3
Figure 3
Effect of calcium ions (A), and surfactant (B), on DrPLA2-V activity. The incubation time with the appropriate agent was for a period of 60 min and the remaining phospholipase activity was evaluated at the optimal conditions.
Figure 4
Figure 4
Effect of organic solvents on DrPLA2-Vstability. Enzyme was incubated with the appropriate agent for 1 h (A) and 2 h (B) and the remaining phospholipase activity was tested at the optimal conditions.
Figure 5
Figure 5
Cytotoxic potency of DrPL2 -V on Lovo, HCT-116, and MDA-MB-231 cells. Cytotoxicity was assessed using the MTT assay by incubating cells for 24 h with various concentrations (25, 50, 100, and 200 μg) of DrPLA2-V.
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