Spectral analysis and catalytic activities of the microsomal mixed-function oxidase system of the sea anemone (phylum: Cnidaria).
Cnidarians are primarily marine organisms with a wide and diverse habitat worldwide. Previous studies from our laboratory demonstrated the presence of proteins in the molecular mass range of 50-60 KDa that were recognized by several antibodies raised against fish cytochromes P450 of the CYP 1, 2, and 3 families in microsomes prepared from the columnar regions of 5 species of sea anemones (Heffernan et al. Mar Environ Res 1996;42:353-357). Pursuant to those studies we report herein results of spectral analyses, NAD(P)H-oxidoreductase and ethoxyresorufin O-dealkylase (EROD) of sea anemone microsomal fractions. The predominant feature in the difference spectrum of dithionite (DTN)-reduced, CO-liganded anemone microsomes was a peak with lambda max of approximately 418 nm, which slowly increased for about 20 min and decreased after about 40 min. A relatively lower amplitude 450 nm peak was attained within 5 min of CO addition and was stable for up to 90 min. The 450 nm peak did not increase concomitant to the decrease in the 418 nm peak suggesting that the latter is not denatured P450. A significantly larger 450 nm peak was attained in CO-difference spectra when DTN was added prior to CO. NADPH-dependent cytochrome c (P450) reductase of the sea anemones was 1.8-3.9 nmol/min/mg protein, which is at the lower end of the range observed in invertebrates. NADH-cytochrome c reductase was 9-25 nmol/min/mg protein, while the NADH-ferricyanide (b5) reductase ranged from 73-232 nmol/min/mg protein. When microsomal EROD activity was measured under conditions in which the reaction was linear with respect to protein concentration, a decrease in fluorescence was typically observed for the initial 15 min of the time course and then increased linearly for up to 90 min; initial velocities were determined from the increasing linear region. NADPH was the preferred cofactor for EROD activity and the NAD(P)H-EROD activity was higher in Anthopleura elegantissima than Anthopleura xanthogrammica. The Bunodosoma cavernata NADPH-EROD activity was barely noted at the detection limit of the assay and NADH-EROD activity was not detected. These results are consistent with a functional P450-dependent MFO system in cnidarians, with characteristics both similar to and unique from other marine invertebrates.