Year-end Sale % OFF 
Abstract
Two full-length cDNA clones (2.2 kilobases) encoding a newly recognized form of mammalian flavin-containing monooxygenase (FMO) have been isolated from independent libraries constructed with mRNA from different rabbits. The cDNAs encode a polypeptide of 533 amino acids which contains two putative pyrophosphate binding domains and a hydrophobic carboxyl terminus characteristic of FMOs. This sequence is 52 and 57% identical to sequences of the rabbit "hepatic" and "pulmonary" FMOs, respectively, and 55% identical to the sequence of "liver form 2" published recently by Ozols (Ozols, J. (1991) Arch. Biochem. Biophys. 290, 103-115). cDNA for the new FMO (FMO 1C1) hybridizes with two species of mRNA, one of 2.6 kilobases and one of about 5.4 kilobases, from liver or kidney, but not lung. Guinea pig, hamster, rat, and mouse all express this form of FMO in liver, kidney, and lung. FMO 1C1 has been tentatively characterized following expression in Escherichia coli. It is inactive with methimazole as substrate but highly active with n-octylamine. The temperature lability, responses to ions and detergent, and pH optimum of FMO 1C1 are similar to values reported for hepatic FMO. Sequence comparisons and analysis of rabbit and human genomic DNA indicate that FMO 1C1, as well as the pulmonary and hepatic FMOs, comprise a single gene family made up of distinct gene subfamilies (A, B,C,D, ... N), each appearing to contain a single gene. A nomenclature, based on these interrelationships and following the same designations used for classifying cytochromes P-450, is proposed.
Highlights
From the Laboratorv of Cellular and Molecular Pharmacology, National InstituotfeEnvironmental Health Sciences, Research Triangle Park, NorthCarolina 27709
Two full-length cDNA clones (2.2 kilobases) encod- second familyof enzymes, the flavin-containingmonooxygening a newlyrecognized form of mammalianflavin- ases (FMOs’: EC 1.14.13.8), is beginning to emerge
Mammalian microsomal monooxygenases havethe capacity arations, and our results indicate that multiple forms of the to metabolizea remarkable variety of exogenous chemicals FMO are present in both tissues[18].To explore further the including many drugs,pesticides, and industrial by-products. possibility that additional forms of the FMO exist, we have
Summary
Isolation of mRNA and Genomic DNA-Total RNA was obtained from liver, kidney, and lung of rabbits(adult male New Zealand White; Dutchland Farms, Denver, PA) by a modification [19] of the methods of Chirgwin et al [20] and Glisin et al [21].Polyadenylated mRNA was isolated by chromatography on oligo(dT)-cellulose [22]. The blots were washed twice with 1X SSC and 0.1% SDS for 30 min at 42 "C and thensubjected to autoradiography for 72 h at -80 "C. Blots that were subsequently hybridized with a second probe were first stripped with a solution of 0.01 x SSC and 0.5% SDS for 20 min at 100 "C. Southern Blot Analysis-Samples of genomic DNA (15 pg) from human placenta and rabbit liver were digested with HindIII, EcoRI, and PstI restriction endonucleases, electrophoresed in agarose gels (0.7%),and transferred to nylon (Nytran) membranes [30].Following transfer, the membranes were rinsed with 5 X SSC for 5 min at 65 "C, baked under vacuum for 2 h at 80 "C, and treated with prehybridization solution for 4 h a t 42 "C. Inall cases protein concentrations were determined by the method of Lowry et al [36]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
