Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens.
Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 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 NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-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.
Ozols
J
J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06032.
D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D005420:Flavoproteins / Q000032:analysis*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000032:analysis; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011817:Rabbits
D005420:Flavoproteins; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases