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Through the use of a number of bioconversion experiments we demonstrated that P450 proteins (CYP1A9 and CYP1C1) from Japanese eel ( Anguilla japonica ) metabolized a number of herbicides and the drug phenacetin. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolites were extracted and analyzed by high-performance liquid chromatography. Proteins CYP1A9 and CYP1C1 metabolized 50 nmol of the drug phenacetin to yield 12.1 and 1.1 nmol of product (acetaminophen), respectively. Further incubation of CYP1A9 with 50 nmol of the herbicides chlorotoluron, diuron, linuron, simazine, or atrazine yielded 16.5, 18.5, 7.3, 1.6, or 0.8 nmol of product, respectively. CYP1C1 also metabolized linuron, diuron, and simazine yield 5.4, 4.6, or 0.7 nmol of product, respectively. Next, polyclonal antibody was isolated by immunizing with two conjugated-peptides (amino acid residues 272–290 and 294–310) of CYP1A9. This antibody did not recognize human CYP1A2 or CYP1C1. Western blotting using the antibody revealed one band in the livers of Japanese eel and tilapia ( Oreochromis niloticus ). Theses results suggest that CYP1A9 and CYP1C1 metabolize herbicides, and that CYP1A9 is an useful biomarker of contamination when detected with this antibody.

Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol red-free condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition. The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound free bilirubin.

To study the effects of urea on the denaturation of myosin B prepared from smooth dogfish, myosin B was kept at 20°C for 10 min with various concentrations (0-3.0M) of urea, and the reduced viscosity was measured as a parameter for structural changes in myosin B. The decrease in the reduced viscosity of myosin B containing urea above 1.0M was similar to that of myosin in the presence of urea, However, the reduced viscosity of F-actin was slightly affected by urea even above 1.0M. The reduced viscosity of myosin B did not recovered to its initial level through the removal of urea at concentrations above 1.0M urea, but that of myosin recovered perfectly through the re-moval of urea at concentrations up to 3.0M urea. In the presence of urea above 1.0M, myosin B irreversibly dissociated into myosin and actin. Therefore, it is concluded that the urea denaturation of myosin B would be attributed to its dissociation into myosin and actin as well as the conforma-tional changes of myosin molecules in the presence of urea above 1.0M. Accordingly, shark myosin B is fairly stable under physiological concentrations of urea below 1.0M.

We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4'-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize.