RESUMO
Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including Km of 90.4 µM and Vmax of 2.63 IU were determined for LOXe. The enzyme with 0.1 nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.
Assuntos
Proteínas de Anfíbios , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Lipoxigenases , Ambystoma mexicanum , Proteínas de Anfíbios/biossíntese , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Animais , Epiderme , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Humanos , Lipoxigenases/biossíntese , Lipoxigenases/genética , Lipoxigenases/isolamento & purificação , Lipoxigenases/farmacologia , Masculino , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Plant growth-promoting rhizobacteria are natural inhabitants of roots, colonize diverse monocot and dicot species, and affect several functional traits such as root architecture, adaptation to adverse environments, and protect plants from pathogens. N,N-dimethyl-hexadecylamine (C16-DMA) is a rhizobacterial amino lipid that modulates the postembryonic development of several plants, likely as part of volatile blends. In this work, we evaluated the bioactivity of C16-DMA and other related N,N-dimethyl-amines with varied length and found that inhibition of primary root growth was related to the length of the acyl chain. C16-DMA inhibited primary root growth affecting cell division and elongation, while promoting lateral root formation and root hair growth and density in Arabidopsis thaliana (Arabidopsis) wild-type (WT) seedlings. Interestingly, C16-DMA induced the expression of the jasmonic acid (JA)-responsive gene marker pLOX2:uidA, while JA-related mutants jar1, coi1-1, and myc2 affected on JA biosynthesis and perception, respectively, are compromised in C16-DMA responses. Comparison of auxin-regulated gene expression, root architectural changes in WT, and auxin-related mutants aux1-7, tir1/afb2/afb3, and arf7-1/arf19-1 to C16-DMA shows that the C16-DMA effects occur independently of auxin signaling. Together, these results reveal a novel class of aminolipids modulating root organogenesis via crosstalk with the JA signaling pathway.