The characterization of aniline hydroxylase in rainbow trout (Salmo gairdneri) liver microsomes

SUMMARY The Characterization of The Activity of Aniline Hydroxylase In Rainbow Trout "Salmo gairdneri" Liver Microsomes The aim in this work is to investigate the abüity of trout to metabolize aniline in liver microsomes of rainbow trout, Salmo gairdneri, cultured in the Eastern Blacksea. It was found that microsomes prepared by centrifiigation were highly capable of catalyzing hydroxylation of aniline to /?-aminophenol. A specific activity of 0.0686 nmol/min/mg protein was found for aniline substrate. Total protein in microsomes was determined to be 28.85 mg/mL. Optimum activity for the liver enzyme was observed in potassium phosphate buffer, pH 7.4, at 25 °C and the rate of aniline hydroxylation was linear with protein concentrations above 1.80 mg microsomal protein. The content of cytochrome P450, one of the mixed function oxidase components, was found as 0.1122 nmol P450/mg protein by carbonmonooxide spectra of dithionite-reduced microsomes using an extinction coefficient of 91 mM-'cm-1. It was seen that NADPH was essential for maximal activity of aniline hydroxylase in liver microsomes. When NADPH and NADH were used together, a little higher activity, 7.7%, was observed with microsomes. In addition a NADPH-generating system almost competes for NADPH. It was observed that the rate of aniline hydroxylation was reached to a V^ value of 5.30 U /mg protein above 0.5 mM aniline concentrations. Lineweaver-Burk and Eadie-Hofstee plots gave almost identical KM and V^ values which are consistent with Michaelis-Menten kinetics. These results also show that a single enzyme is responsible for hydroxylation of aniline in trout liver microsomes. Finally, the effects of several metal ions on trout liver aniline hydroxylase were investigated, 0.1 mM concentrations of Mg+2, Cd+2 and Fe+3 had almost similar stimulatory effect on the activity. Cu+2 dramatically increased the activity of the enzyme. In contrast, Ni+2 and Hg+2 being two of the most toxic metal ions inhibited the rate of aniline hydroxilation by 25% at 1 mM concentrations. Further in vitro and in vivo experiments should be done in order to get more detailed information on the mechanism, structure and function of enzymatic hydroxylation systems offish. Key Words: Salmo gairdneri. Liver Microsomes, Aniline Hydroxylase, Cytochrome P450, Aniline, Activity, Characterization VI