Kinetic characterization of the catalysis of "activated" cyclophosphamide (4-hydroxycyclophosphamide/aldophosphamide) oxidation to carboxyphosphamide by mouse hepatic aldehyde dehydrogenases.
A spectrophotometric assay was developed and utilized to directly characterize aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide to carboxyphosphamide by soluble and solubilized particulate fractions prepared from mouse liver homogenates. Vmax values of 3310 and 1170 nmol/min/g liver were obtained for the soluble and solubilized particulate fractions respectively. Km values were 22 and 84 microM respectively. Alkaline pH optimums were observed in each case. Aldehyde dehydrogenase-catalyzed oxidation of aldophosphamide by the soluble fraction was markedly more temperature responsive. Catalysis of aldophosphamide and acetaldehyde or benzaldehyde oxidation was apparently by the same isozyme(s) in the soluble fraction. Similarly, low Km (acetaldehyde/benzaldehyde) and high Km (acetaldehyde/benzaldehyde) isozymes each apparently catalyzed the oxidation of aldophosphamide in the solubilized particulate fraction. Our findings suggest that (1) oxidation of aldophosphamide to carboxyphosphamide by mouse liver is catalyzed largely by the predominant aldehyde dehydrogenase isozyme present in the soluble fraction (cytosol) of this tissue, and (2) isozymes that catalyze aldophosphamide oxidation are not different from those that catalyze the oxidation of acetaldehyde and benzaldehyde, though the relative contribution of each isozyme within the solubilized particulate fraction to the catalysis of aldophosphamide oxidation remains to be determined.
Manthey
C L
CL
Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Sladek
N E
NE
D000444:Aldehyde Dehydrogenase / Q000032:analysis / Q000494:pharmacology*; D000818:Animals; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D007527:Isoenzymes / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008811:Mice, Inbred DBA; D010084:Oxidation-Reduction; D010752:Phosphoramide Mustards / Q000378:metabolism*
D007527:Isoenzymes; D010752:Phosphoramide Mustards; C012358:4-hydroxycyclophosphamide; C006446:aldophosphamide; D003520:Cyclophosphamide; D000444:Aldehyde Dehydrogenase