Solubilization and partial purification of two forms of cytochrome P-450 from trout liver microsomes

Abstract

1. 1. Liver microsomal cytochrome P-450 content, NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethylmorphine N- demethylase activities of rainbow trout, Salmo gairdneri, were found to be 0.16 ( n = 10) nmol P-450/mg protein, 38 ( n = 5) units/mg protein, 0.04 ( n = 4) nmol p- aminophenol/min/mg protein and 0.174 ( n = 3) nmol formaldehyde/min/mg protein, respectively. 2. 2. Trout liver cytochrome P-450 was solubilized by treatment of microsomes with sodium cholate. Chromatography on DEAE-cellulose column yielded two distinct cytochrome P-450 fractions from solubilized microsomes. Cytochrome P-450-I was eluted with Emulgen 913-containing buffer. Application of 0.08 M KCl in Emulgen 913-containing buffer to the DEAE-cellulose column eluted cytochrome P-450-II fraction. Cytochrome P-450-I was further purified on hydroxylapatite column. 3. 3. CO-difference spectrum of dithionite-reduced cytochrome P-450-I gave a peak at 449 nm while the similar spectrum of cytochrome P-450-II showed a maximum absorbance at 451 nm. Monomer molecular weights of cytochrome P-450-I and cytochrome P-450-II were determined by sodium dodecyl sulfate gel electrophoresis and found to be 56,000 and 48,500, respectively.