Purification and characterization of guinea pig liver morphine 6-dehydrogenase.

Abstract

Morphine 6-dehydrogenaq which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%.The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing.The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively.The enzyme utilizes both NAD and NADP as a cofactor, and the K , values were 0.12 mM for NAD and 0.42 mM for NADP.The V,, values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP).The K , values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP).The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates.The enzyme was inactivated by removal of 2mercaptoethanol from the enzyme solution.The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol.Phenylarsine oxide and CdClz (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding.These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site.Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra.It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol.Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2- mercaptoethanol to morphinone.The metabolism of morphine has been studied extensively in many animal species.Morphine is metabolized mainly via glucuronide conjugation and N-demethylation (1-3).Dihydromorphinone was identified as a new metabolite of morphine in the acid-hydrolyzed urines of mouse, rat, rabbit, guinea pig, cat, and monkey (4, 5).This metabolite possibly occurred by two-step reactions: the dehydrogenation of the 6hydroxyl group and then the hydration of the 7,8-unsaturated bond and vice versa.The enzymes participating in these Guinea Pig Liver Morphine 6-Dehydrogenase TABLE I Summary of the purification of morphine 6-dehydrogenase from guinea pig liver The enzyme was assayed with NAD as described under "Experimental Procedures."Fraction Total Total Specific Purifi-Yield protein activity activity cation mg units milliunits/ -fold % mg Soluble" 13,582 16.8 1.2 1 100 (NH,),SO, 5,937 14.8 2.