1361
J. Med. Chem. 1985,28, 1361-1365 absorption of the mixture was monitored a t 265 nm on a Cary Model 14 recording spectrophotometer for 2 h. After filtration through membrane cones (Amicon Centriflo F 25, Amicon Corp.) and concentration, the mixtures were analyzed for substrate and product by HPLC using the conditions described above. Control reactions were conducted with adenosine and ara-A. (b) 5'-Adenylic Acid Deaminase (Rabbit Muscle). A spectrophotometric assay, similar to that described for adenosine deaminase, above, was used. The enzyme (0.1 mL, 10.0 unita/mL), in 0.05 M phosphate buffer (pH 7.5), was added at room temperature to a solution of IC and Id (0.130 Fmol) in 0.05 M phosphate buffer (pH 7.5, 2.9 mL). The absorption of the mixture was monitored at 265 nm for 2 h. Control reactions were conducted with AMP and araAMP. 3. Cytochrome P-450 Dependent Mixed-Function Oxidases. BDF, mice were injected intraperitoneally with sodium phenobarbital (75 mg/kg) for 4 consecutive days. The animals were killed 24 h after the last injection, and the livers were excised. A 33% liver homogenate in 0.05 M Tris-HCl-O.15 M KCl-O.01 M MgC12 buffer (pH 7.4) was centrifuged at lOOOOg for 20 min at 4 "C. The supematant fraction was aspirated and centrifuged at 105OOOg for 60 min at 4 "C. The microsomal pellet was washed by resuspension in the original volume of buffer containing 0.01 M EDTA and was then resedimented at 105OOOg for 30 min. The final pellet was reconstituted in the Tris-KC1-MgC12 buffer such that each mL of suspension contained microsomes from 0.33-g wet weight of liver. Each incubation mixture contained cyclic
5'-nucleotide (1mM), NADH (0.4 mM), glucose 6-phosphate (5.0 mM), glucose &phosphate dehydrogenase (0.6 unit/mL), and 0.25 mL of microsomal suspension in a total volume of 1.25 mL. After 1h at 37 OC, the incubates were transferred to Amicon Centriflo CF 25 membrane cones (Amicon Corp., Lexington, MA) and centrifuged at 2000 rpm (
