Carboxylesterases and arylacetamide deacetylase comparison in human A549, H460, and H727 pulmonary cells
Carboxylesterases and arylacetamide deacetylase comparison in human A549, H460, and H727 pulmonary cells
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DOI：
10.1016/j.lfs.2021.119486
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发表时间：
2021-04-18
期刊：
影响因子：
6.1
通讯作者：
Longo, Vincenzo
中科院分区：
文献类型：
--
作者：
Gabriele, Morena;Puccini, Paola;Longo, Vincenzo
目的：人羧酸酯酶（CES）和芳基乙酰胺脱乙酰酶（AADAC）是丝氨酸酯酶，催化许多含有酯、酰胺、硫酯或乙酰基的化合物的水解。本研究的目的是调查的存在，动力学参数，和抑制A549，H460，和H727肺细胞中的活细胞和S9 fractions.Materials和方法：的p-nitrophenyl acetate（pNPA）和4-methylumbelliferyl acetate（4-MUA）被用作非选择性酯酶底物，而非那西丁作为选择性AADAC底物。主要发现：AADAC基因在A549和H460细胞中被检测到，而在S9相关组分中未检测到芳基酯酶活性。此外，所有肺细胞都不同程度地表达CES 1和CES 2，并且酶活性彼此相当重叠。所有的酶都表现出典型的米氏饱和曲线，关于4-MUA，在活细胞和S9组分中发现了相似的Km值。相反，与S9组分水解pNPA相关的动力学参数显著低于活细胞中检测到的参数。抑制研究表明，4-MUA水解受到双-对硝基-苯基磷酸盐和苯甲磺酰氟的抑制作用大于洛哌丁胺;相反，pNPA水解抑制作用有限，在活细胞和S9组分中获得了相似的抑制曲线。羧酸酯酶的存在肯定证实了细胞bioimaging.Significance：这些研究结果增加了信息，酯酶的知识在肺细胞，可用作毒理学和药理学研究的体外模型。
Aims: Human carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) are serine-esterase enzymes catalyzing the hydrolysis of many compounds containing esters, amides, thioesters, or acetyl groups. This study aimed to investigate the presence, kinetic parameters, and inhibition of CES1, CES2, and AADAC in A549, H460, and H727 pulmonary cells in both living cells and S9 fractions.Materials and methods: The p-nitrophenyl acetate (pNPA) and 4-methylumbelliferyl acetate (4-MUA) were used as non-selective esterase substrates, whereas phenacetin as selective AADAC substrate. CESs activities were also investigated in living cells by cellular bioimaging using selective fluorescent probes.Key findings: AADAC gene was detected in A549 and H460 cells; nevertheless, arylesterase activity was not found in relative S9 fractions. Besides, CES1 and CES2 were expressed to a different extent by all lung cells, and enzymatic activities were quite overlapping each other. All enzymes exhibited a typical Michaelis-Menten saturation curve and, regarding 4-MUA, similar Km values were found in both living cells and S9 fractions. Conversely, kinetic parameters relative to the pNPA hydrolysis by S9 fractions were significantly lower than those detected in living cells. Inhibition studies revealed that 4-MUA hydrolysis was inhibited by bis-p-nitro-phenyl phosphate and phenylmethanesulfonyl fluoride more than loperamide; on the contrary, pNPA hydrolysis inhibition was limited with similar inhibition profiles being obtained in both living cells and S9 fractions. The presence of carboxylesterases was definitely confirmed by cellular bioimaging.Significance: These findings add information to esterase knowledge in pulmonary cells that could be used as in vitro models for toxicological and pharmacological studies.