Abstract
DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.
Highlights
Polyamines are small molecular weight, positively charged compounds that are ubiquitous in all living cells
Since de novo polyamine synthesis in both P. carinii and mammalian cells is blocked by DFMO, these data led to the hypothesis that polyamine catabolism is not regulated in P. carinii, which would cause this pathogen to be vulnerable to compounds that interfere with polyamine synthesis [8]
One critical experiment that had been done previously with cells isolated from infected animals was repeated with cultured cells for comparison and for validation of other results obtained with cultured cells; i.e. a study was made of the rate of polyamine depletion in cultured cells in which polyamine synthesis was blocked by DFMO
Summary
The final step is removal of the acetylated aminopropyl group by polyamine oxidase (PAO), a FAD-dependent enzyme which produces 3-acetoamidopropanal and either spermidine or putrescine from N-acetylated spermine or spermidine, respectively. Down-regulation of SSAT prevents polyamine depletion in mammalian cells when polyamine synthesis is blocked by DFMO [11]. Since de novo polyamine synthesis in both P. carinii and mammalian cells is blocked by DFMO, these data led to the hypothesis that polyamine catabolism is not regulated in P. carinii, which would cause this pathogen to be vulnerable to compounds that interfere with polyamine synthesis [8]. The work reported here supports that hypothesis by demonstrating that SSAT and PAO activities are not reduced when cellular polyamine concentrations decrease due to inhibition of ornithine decarboxylase by DFMO. As internal polyamine concentrations decrease as a result of polyamine catabolism, excretion from the cell further reduces the polyamine pool
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