N-hydroxylation of benzamidine to benzamidoxime by a reconstituted cytochrome P-450 oxidase system from rabbit liver: involvement of cytochrome P-450 IIC3.
Previous investigations have provided evidence for the participation of the cytochrome P-450 (P-450) enzyme system in the established N-hydroxylation of benzamidine to benzamidoxime by microsomal fractions from rabbit liver homogenates. In the present investigation, a representative mixture of P-450 isoenzymes was first isolated from the livers of untreated rabbits and then, together with purified NADPH-P-450 reductase, successfully used in a reconstituted enzyme system for the N-hydroxylation of benzamidine. In order to identify the participating isoenzyme, the P-450 mixture was separated by preparative high performance liquid chromatography on an anion exchange column. A P-450 fraction was obtained that was able to transform benzamidine with a specific activity > 3-fold higher than that of the P-450 mixture. The electrophoretic and spectral properties, as well as the inhibition by monoclonal antibodies against P-450 IIC3, show that the isolated P-450 fraction must consist of one or more variants of the isoenzyme P-450 IIC3. By means of reconstitution experiments with highly purified variants of P-450 IIC3 from rabbit liver and with purified variants of P-450 IIC expressed by recombinant Escherichia coli, the participation of the two variants P-450 IIC3 (6 beta H) and P-450 IIC3 (6 beta L) in the N-hydroxylation of benzamidine was unequivocally confirmed.
Clement
B
B
Pharmazeutisches Institut, Christian Albrechts Universität, Kiel, Germany.
Jung
F
F
Pfunder
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D001550:Benzamidines / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006900:Hydroxylation; D007527:Isoenzymes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D001550:Benzamidines; D007527:Isoenzymes; C053561:benzamidoxime; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C032157:benzamidine