Liver microsomes contain two distinct NADPH-monooxygenases with NH 2-terminal segments homologous to the flavin containing NADPH monooxygenase of [formula omitted][formula omitted

Abstract

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as form 1 and form 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2′, 5′-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH 2- terminus of form 1 is blocked, whereas the NH 2-terminus of form 2 is homologous to the NADPH-p-hydroxybenzoate hydrolase from Pseudomonas fluorescens . The latter and the form 2 enzyme share 11 identical residues in the NH 2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.