Abstract
N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.
Highlights
Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, HzOz, and a polyamine analog with free terminal amines
We reported previously that micromolar concentrations of bis(benzyl)polyamine analogs inhibited the growth of Plusmodium falciparum, a human malaria parasite, in vitro and, in combination with cu-difluoromethylornithine, an irreversible inhibitor of polyamine biosynthesis, cured Plasmodium berghei infections in mice [1]
We found that the bis(benzyl)polyamine analogs were potent inhibitors of rat hepatoma (HTC) cell growth in vitro [2]
Summary
Preparation of Rat liver Polyamine Oxidase-Polyamine oxidase was purified from rat liver using modifications of techniques described by Hiilttii [7]. Four fractions (24 ml total) which contained the highest polyamine oxidase activity were pooled and concentrated to 7 ml with an Amicon ultrafiltration cell equipped with a PM-10 membrane. The columns were equilibrated and polyamine oxidase (1.4.mg protein sample), purified through the DEAE-cellulose step, was eluted with 20 mM potassium phosphate (pH 6.5) containing 100. Leukocytes were removed from the blood by loading whole blood onto a 2-ml column of sulfoethyl cellulose (SERVA) which had been equilibrated previously with a solution of 10 mM potassium phosphate (pH 7.5) containing MgC12, 138 mM NaCl, and 0.4 mM EDTA [17]. The final red cell pellet was extracted with 2 volumes of 0.4 M perchloric acid and the polyamine analogs were measured by HPLC as detailed above. Other analytical reagents, including N’-acetylspermidine and N’-acetylspermine, were purchased from Sigma
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