Characterization of ethoxyresorufin O-deethylase activity (EROD) in oyster Crassostrea brasiliana

详细信息    查看全文

• 作者：Marilia N. Siebert^a ; Jacó ; J. Mattos^b ; Clei E. Piazza^a ; Daí ; na de Lima^a ; Carlos Henrique A.M. Gomes^c ; Clá ; udio M.R. de Melo^c ; Afonso C.D. Bainy^a ; ^{afonso.bainy@ufsc.br}
• 关键词：7-ER ; 7-ethoxyresorufin ; AHRs ; aryl hydrocarbon receptors ; ANF ; α-naphthoflavone ; CYP ; cytochrome p450 ; DMSO ; dimethyl sulfoxide ; DTT ; dl-dithiothreitol ; ELP ; ellipticine ; EROD ; 7-ethoxyresorufin O-deethylase activity ; FRF ; furafylline ; NADH ; β-nicotinamide adenine dinucleotide reduced ; NADPH ; β-nicotinamide adenine dinucleotide 2&prime ; -phosphate reduced ; PAH ; polycyclic aromatic hydrocarbon ; PHE ; phenanthrene ; PMSF ; phenylmethanesulfonyl fluoride
• 刊名：Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：203
• 期：Complete
• 页码：115-121
• 全文大小：744 K
• 卷排序：203

文摘

Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30 °C and 7.4, respectively. EROD apparent Km (Kmapp) was 4.32 μM for gills and 5.56 μM for digestive gland. EROD Vmax was 337.3 fmol·min− 1·mg of protein− 1 in gills and 297.7 fmol·min− 1·mg of protein− 1 in digestive gland. Compared to other bivalves, a higher Kmapp and a lower Vmax was found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100 μM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.