Inhibition of Adenosine Deaminase by a Metabolite of the Nephrotoxic Drug,Puromycin Aminonucleoside. ∗

Abstract

Administration of the aminonucleoside of puromycin (PA)‡ to the rat produces a ne-phrotic syndrome characterized by proteinuria, hypoalbuminemia, hypercholesterolemia, edema, and ascites(l-4). The principal necleo-side metabolite in 8-hour rat urine after PA administration is the monodemethylated analog, MMPA(5). Previous studies by Cory and Suhadolnik(6) showed that the didemethylated analog of PA, APA, is a substrate for adenosine deaminase (E. C. 3.5.4.4) and that N⁶-methyladenosine is an inhibitor of the enzyme. Model studies by Schaeffer et al(7) suggested that MMPA would be an inhibitor of adenosine deaminase. The present studies demonstrate that MMPA does indeed inhibit adenosine deaminase.

Materials and methods. Calf intestinal mucosal adenosine deaminase, Type II, and N⁶-methyladenosine were purchased from the Sigma Chemical Co.

The monodemethylated analog of PA, was prepared biosynthetically by in of PA with fortified rat liver micro according to a modification of the proof Axelrod(8). P,4 (55.6 mg) was in at 25′ with the microsomes from 10 of liver together with the requisite cofactors (8) for 4 hours with stirring. The reaction stopped by heating in a boiling water for 10 minutes, and the aminonucleosides by ion-exchange chromatography according to Wilson et al(9). The aminonu were separated by chromatography Whatman 3 MM paper with n-propanol-1 KH40H (75: 25, v/v), corresponding to solvent system “C”(9). The MM band eluted with water was repurified by chromatography on Silica Gel HF with a chloroform-methanol-2.5 ammonia (150: 100: 15, v/v), (CMN) Solvent. After elution from the thin-layer plate with water the MMPA was refractionated on the ion-exchange iysteni to re-move some impurities picked up from the thin-layer absorbent.