CHARACTERIZATION AND PARTIAL PURIFICATION OF THE RAT AND HUMAN ENZYME SYSTEMS ACTIVE IN THE REDUCTION OF N-HYDROXYMELAGATRAN AND BENZAMIDOXIME
Authors:
- Yvonne HofmannYHYvonne Hofmann
- Åsa NordlingÅNÅsa Nordling
- Xue-Qing LiXLXue-Qing Li
- Sabina NiveliusSNSabina Nivelius
- Tommy B. AnderssonTATommy B. Andersson
- Magnus Ingelman-SundbergMIMagnus Ingelman-Sundberg
- Inger JohanssonIJInger Johansson
Open Access
- 1 April 2005
- journal article
- Published by Elsevier in Drug Metabolism and Disposition
- Vol. 33 (4) , 570-578
Abstract
The enzymic basis for intracellular reduction of N-hydroxylated amidines to their corresponding amidines, and hydroxylamines to their corresponding amines, is unknown. The hydroxylated amidines can be used as prodrug moieties, and an understanding of the enzyme system active in the reduction can contribute to more efficient drug development. In this study, we examined the properties of this enzyme system using benzamidoxime and N-hydroxymelagatran as substrates. In rats and humans, the hepatic enzyme system was localized in mitochondria as well as in microsomes, using preferably NADH as cofactor. Potassium cyanide, N-methylhydroxylamine, p-hydroxymercuribenzoate, and desferrioxamine were efficient inhibitors, whereas typical cytochrome P450 (P450) inhibitors were ineffective. In rats, the highest specific activity was found in liver, adipose tissue, and kidneys, whereas in humans, the specific activity in the preparations of adipose tissue examined was lower. A sex difference was observed in rat liver, where 4-fold higher activity was seen in microsomes from female rats. No gender differences were present in any other tissue investigated. Partial purification of the hepatic system was achieved using polyethylene glycol fractionation followed by Octyl Sepharose chromatography at low detergent concentrations, whereas the enzyme was denatured after complete solubilization. The unique appearance of the enzyme activity in adipose tissue, together with the cyanide sensitivity and the failure of typical P450 inhibitors to impede the reaction, indicates that the enzyme system active in reduction of benzamidoxime and N-hydroxymelagatran formation is not of cytochrome P450 origin, but likely consists of an NADH-dependent electron transfer chain with a cyanide-sensitive protein as the terminal component.Keywords
This publication has 29 references indexed in Scilit:
- Characterization of in Vitro Biotransformation of New, Orally Active, Direct Thrombin Inhibitor Ximelagatran, an Amidoxime and Ester ProdrugDrug Metabolism and Disposition, 2003
- REDUCTION OFN-HYDROXYLATED COMPOUNDS: AMIDOXIMES (N-HYDROXYAMIDINES) AS PRO-DRUGS OF AMIDINESDrug Metabolism Reviews, 2002
- Reduction of Amphetamine Hydroxylamine and Other Aliphatic Hydroxylamines by Benzamidoxime Reductase and Human Liver MicrosomesChemical Research in Toxicology, 2000
- Effects of Melagatran, a New Low-molecular-weight Thrombin Inhibitor, on Thrombin and Fibrinolytic EnzymesThrombosis and Haemostasis, 1998
- Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime ReductaseJournal of Biological Chemistry, 1997
- A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomesArchives of Toxicology, 1996
- Covalent Binding of Sulfamethoxazole Reactive Metabolites to Human and Rat Liver Subcellular Fractions Assessed by Immunochemical DetectionChemical Research in Toxicology, 1996
- A high-yield preparative method for isolation of rat liver mitochondriaAnalytical Biochemistry, 1977
- The hydroxylamine reductase of mitochondriaArchives of Biochemistry and Biophysics, 1969
- Reduction of hydroxylamine by rat liver mitochondriaArchives of Biochemistry and Biophysics, 1968