Isolation and structure of a third form of liver microsomal flavin monooxygenase.
Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent oxygenation of nucleophilic nitrogen, sulfur, and phosphorous atoms in various drugs, pesticides, and xenobiotics. Two forms of this enzyme have been isolated and characterized from rabbit liver microsomes [Ozols, J. (1989) Biochem. Biophys. Res. Commun. 163, 49-55]. The isolation and the structure of a third isoform (FMO3) is presented here. The isolation procedure for FMO3 included solubilization of liver microsomes with cholate, poly(ethylene glycol) precipitation, chromatography on anion- and cation-exchange and hydroxyapatite columns in the presence of nonionic detergents and glycerol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, FMO3 exhibited a distinct, single band with a M(r) higher than those of FMO1 and FMO2. FMO3 copurified with a polypeptide complex of high FMO activity. This complex consisted of three polypeptides, named FMO3, FMO1a, and FMO2a. The column chromatographic behavior of FMO1 a and 2a was distinct from that of FMO1 and 2. The electrophoretic mobility of FMO1a was identical to that of FMO1. Automated sequence analysis of this polypeptide complex indicated the presence of only one predominant peptide with an open N-terminus. The derived N-terminal amino acid sequence of some 20 residues was identical to the N-terminus of FMO2. The FMO complex, however, did not contain a polypeptide corresponding to the electrophoretic mobility of FMO2. The N-terminus of FMO3 was blocked by an acetyl residue. Automated Edman degradation of peptides obtained from chemical and enzymatic digests established the amino acid sequence of some 514 residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Ozols
J
J
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305.
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D003488:Cyanogen Bromide; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012995:Solubility; D014357:Trypsin / Q000378:metabolism
D003902:Detergents; D007527:Isoenzymes; D010446:Peptide Fragments; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D010450:Endopeptidases; D014357:Trypsin; D003488:Cyanogen Bromide