Identification of N-acetylspermidine in Leishmania amazonensis

Rojas-Chaves, Miguel; Hellmund, Claudia; Walter, Rolf D.

doi:10.1016/0166-6851(95)02520-0

Cited by 4 publications

(8 citation statements)

References 12 publications

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“…The derivatized polyamines were recovered with two ethyl acetate extractions, and the organic layers were pooled and dried. Samples were resuspended in 200 l of 95% methanol/5% acetic acid, and polyamines were analyzed by high performance liquid chromatography on a Beckman system equipped with a C 18 reversed phase column (Bio-Rad) as described previously (25). Relative fluorescence was measured on a Shimadzu RF-535 fluorescence high performance liquid chromatography monitor at excitation and emission wavelengths of 365 and 485 nm, respectively.…”

Section: Materials Chemicals and Reagents-[cooh-mentioning

confidence: 99%

“…The trichloroacetic acid was extracted with ethyl acetate as reported previously (24), and the samples were dried on a Speed Vac concentrator. Samples were pre-column derivatized with dansyl chloride using previously reported protocols (25), except that the sample volumes were 100 l. 100 l of 25% proline were added to scavenge excess dansyl chloride. The derivatized polyamines were recovered with two ethyl acetate extractions, and the organic layers were pooled and dried.…”

Section: Materials Chemicals and Reagents-[cooh-mentioning

confidence: 99%

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Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Jiang¹,

Roberts²,

Jardim³

et al. 1999

Journal of Biological Chemistry

115

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A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of ⌬odc cells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The ⌬odc L. donovani exhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the ⌬odc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.Polyamines are cationic compounds that play essential roles in cell proliferation, differentiation, and macromolecular synthesis (1-3). Ornithine decarboxylase (ODC) 1 catalyzes the conversion of ornithine to putrescine (1,4-diaminobutane) and is the initial and rate-limiting enzyme in polyamine biosynthesis in most organisms (4). The ODC enzyme of protozoan parasites is a novel therapeutic target, because D,L-␣-difluoromethylornithine (DFMO; eflornithine), an irreversible inhibitor of ODC (5), exhibits notable efficacy against the central nervous system phase of African sleeping sickness caused by Trypanosoma brucei gambiense (3, 6). DFMO is also active against T. b. rhodesiense and T. congolense in murine models and has proven effective against other genera of protozoan parasites in vivo and in vitro, including Plasmodia (7), Giardia (8), and Leishmania (9). DFMO has been shown to induce a lethal polyamine depletion in both T. brucei (10) and L. donovani (9), the etiologic agent of visceral leishmaniasis, and toxicity to both species is ameliorated by polyamine addition (3, 9).The ability of trypanosomatids to undergo a very high frequency of homologous recombination allows the disruption of chromosomal loci with transfected drug resistance cassettes (11,12) and permits a direct test of gene function. This enables the creation of conditionally lethal parasite strains whose survival and ability to propagate are dependent upon the provision of compounds that can ameliorate the consequences of the genetic lesion. This genetic approach is predicated on the availability of c...

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Section: Materials Chemicals and Reagents-[cooh-mentioning

confidence: 99%

Section: Materials Chemicals and Reagents-[cooh-mentioning

confidence: 99%

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Jiang¹,

Roberts²,

Jardim³

et al. 1999

Journal of Biological Chemistry

115

View full text Add to dashboard Cite

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“…The recent demonstration of N-acetylspermidine at a quite low albeit significant, concentration in Leishmania amazonensis promastigotes (Rojas-Chaves et al 1996) suggests that N-acetylspermidine may be a degradation or excretory product of excessive polyamines as described in mammals (Seiler 1987). The occurrence of Nacetylated polyamines has also been reported in helminths; N 1-and NS-acetylspermidine have been identified in the trematodes Fasciola hepatica and Paragonimus uterobilateralis as well as in the spent media of maintained flukes (Aisien and Walter 1992;unpublished data).…”

Section: Discussionmentioning

confidence: 99%

“…In Leishmania, putrescine and spermidine represent the major components of the polyamines, whereas the concentration of spermine is either much lower or questionable (Bachrach et al 1979;Morrow et al 1980;Coombs and Sanderson 1985;Keithly and Fairlamb 1988;Balafia-Fouce et al 1991;Gonzfilez et al 1991). In addition to these polyamines, N j-acetylspermidine has recently been detected in promastigotes of L. amazonensis (Rojas-Chaves et al 1996). In mammals, N-acetylputrescine and N 1-and N 8-acetylspermidine are terminal catabolites, which are removed from cells and found as urinary constituents (Seilet 1987).…”

Section: Introductionmentioning

confidence: 99%

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Polyamine N-acetyltransferase in Leishmania amazonensis

1996

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N-Acetyltransferase, which is suggested to be responsible for the production of N1-acetylspermidine in Leishmania amazonensis and to be involved in the process of inactivation and degradation of excessive polyamines, was partially purified and characterized. Among the substrates tested, sym-norspermidine, sym-norspermine, and 1,3-diaminopropane had the highest reaction rates, but the naturally occurring polyamines spermine and spermidine were also acetylated at considerable rates, whereas putrescine was a poor substrate. The Michaelis constants (Km values) for spermine and spermidine were 0.66 and 3.3 mM, respectively. The Km value for acetylcoenzyme A (acetyl-CoA) was determined to be 34 microM. CoA inhibited the reaction in a competitive manner; the inhibition constant was 5 microM. The enzyme showed an apparent relative molecular mass of 35,000.

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Leishmania major thialysine N^ε‐acetyltransferase: Identification of amino acid residues crucial for substrate binding

Lüersen

2005

FEBS Letters

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Thialysine N e -acetyltransferases and spermidine/ spermine N-acetyltransferases (SSAT) are closely related members of the GCN5-related N-acetyltransferase superfamily. Accordingly, a putative orthologue from the human protozoan parasite Leishmania major exhibits an almost equal similarity to human SSAT and thialysine N e -acetyltransferase. Characterisation of the recombinantly expressed L. major protein indicated that it represents a thialysine N e -acetyltransferase, preferring thialysine (S-aminoethyl-L L-cysteine) and structurally related amino acids as acceptor molecules. The known thialysine N e -acetyltransferases contain five conserved amino acid residues that are replaced in SSAT sequences. Kinetic analyses of the respective recombinant mutant proteins suggest that Ser 82 and Thr 83 of L. major thialysine N e -acetyltransferase are key residues for acceptor binding. In addition, the conserved Leu 130 is tentatively involved in specific interaction with the sulphurcontaining side chain of thialysine. The presence of these three amino acid residues is suggested to be a means by which thialysine N e -acetyltransferases can be distinguished from SSAT sequences.

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Identification of N-acetylspermidine in Leishmania amazonensis

Cited by 4 publications

References 12 publications

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Polyamine N-acetyltransferase in Leishmania amazonensis

Leishmania major thialysine N^ε‐acetyltransferase: Identification of amino acid residues crucial for substrate binding

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Identification of N-acetylspermidine in Leishmania amazonensis

Cited by 4 publications

References 12 publications

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

Polyamine N-acetyltransferase in Leishmania amazonensis

Leishmania major thialysine Nε‐acetyltransferase: Identification of amino acid residues crucial for substrate binding

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Product

Resources

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Leishmania major thialysine N^ε‐acetyltransferase: Identification of amino acid residues crucial for substrate binding