[26] Trypanothione and N1-glutathionylspermidine:Isolation and determination

Krauth‐Siegel, R. Luise; Jacoby, E.M.; Schirmer, R. Heiner

doi:10.1016/0076-6879(95)51131-8

Cited by 17 publications

(11 citation statements)

References 16 publications

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“…To measure TRYR, extracts from exponentially growing cells (typically less than 1 × 10 6 cells ml −1 ) were prepared by freezing and thawing in 100 mM Tris‐Cl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol (DTT), followed by centrifugation at 10 000 g in a microfuge (approximately 5 min). TRYR activity was assayed spectrophotometrically by monitoring the NADPH‐dependent reduction of trypanothione disulphide at 340 nm in the same buffer (modified according to Krauth‐Siegel et al ., 1995). Activity was normalized to control cell lines (449), and analyses were performed in duplicate for three independent experiments.…”

Section: Methodsmentioning

confidence: 99%

Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress

Krieger

Schwarz

Ariyanayagam

et al. 2000

Molecular Microbiology

335

243

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SummaryIn Kinetoplastida, trypanothione and trypanothione reductase (TRYR) provide an intracellular reducing environment, substituting for the glutathione±glu-tathione reductase system found in most other organisms. To investigate the physiological role of TRYR in Trypanosoma brucei, we generated cells containing just one trypanothione reductase gene, TRYR, which was under the control of a tetracycline-inducible promoter. This enabled us to regulate TRYR activity in the cells from less than 1% to 400% of wild-type levels by adjusting the concentration of added tetracycline. In normal growth medium (which contains reducing agents), trypanosomes containing less than 10% of wild-type enzyme activity were unable to grow, although the levels of reduced trypanothione and total thiols remained constant. In media lacking reducing agents, hypersensitivity towards hydrogen peroxide (EC 50 3.5 mM) was observed compared with the wild type (EC 50 223 mM). The depletion of TRYR had no effect on susceptibility to melarsen oxide. The infectivity and virulence of the parasites in mice was dependent upon tetracycline-regulated TRYR activity: if the trypanosomes were injected into mice in the absence of tetracycline, no infection was detectable; and when tetracycline was withdrawn from previously infected animals, the parasitaemia was suppressed.

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Section: Methodsmentioning

confidence: 99%

Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress

Krieger

Schwarz

Ariyanayagam

et al. 2000

Molecular Microbiology

335

243

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“…Controls were used without the analogs. To be sure that NADPH consumption was associated to TR activity, an assay was carried out in which it was added an excess (500 mM and 1000 mM) of oxidized glutathione (GSSG), as described before [9,13]. The reduced trypanothione T (SH) 2 formed will react with the excess of GSSG, producing more oxidized trypanothione T(S) 2 and increasing the NADPH consumption, according to the scheme:…”

Section: Tr Activity Assaymentioning

confidence: 99%

Leishmania amazonensis trypanothione reductase: Evaluation of the effect of glutathione analogs on parasite growth, infectivity and enzyme activity

Castro‐Pinto

Lima

Cunha

et al. 2007

Journal of Enzyme Inhibition and Medicinal Chemistry

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Department of Chemistry, UFRJ, Rio de Janeiro, Brazil Abstract Trypanothione reductase (TR) is a major enzyme in trypanosomatids. Its substrate, trypanothione is a molecule containing a tripeptide (L-glutamic acid-cysteine-glycine) coupled to a polyamine, spermidine. This redox system (TR/Trypanothione) is vital for parasite survival within the host cell and has been described as a good target for chemotherapy anti-Leishmania. The use of tripeptides analogs of glutathione would result in a decrease in trypanothione synthesis and as a consequence in TR activity. In this work, besides the enzyme potential inhibition, it also evaluated the influence of those analogs on parasite growth and on its infective capacity. The results showed a significant effect on parasite growth and infectivity and in addition TR activity was highly inhibited. These results are very promising, suggesting a potential use of those analogs as therapeutic drugs against experimental diseases caused by trypanosomatids.

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“…

Tryparedoxin I is a new member of the thioredoxin superfamily of proteins isolated from the trypanosomatid Crithidia fasciculata [7], an insect-pathogenic trypanosomatid parasite commonly used as a model organism to study human pathogens causing important tropical diseases such as African sleeping sickness (Trypanosoma brucei gambiense, T. b. rhodiense), Chagas diseases (Trypanosoma cruzi) and the various forms of Leishmaniasis (Leishmania species).In C. fasciculata tryparedoxin I is a constituent of a unique metabolic pathway reducing hydroperoxides at the expense of NADPH [7]. Its active site containing a disulfide bridge formed by a characteristic WCPPCR motif is specifically reduced [2] by trypanothione, a bis-glutathionyl derivative of spermidine, found in the parasitic Kinetoplastida [3]. Reduced trypanothione is regenerated from its cyclic disulfide form by the NADPH-dependent flavoprotein trypanothione reductase [3].

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mentioning

confidence: 99%

“…Its active site containing a disulfide bridge formed by a characteristic WCPPCR motif is specifically reduced [2] by trypanothione, a bis-glutathionyl derivative of spermidine, found in the parasitic Kinetoplastida [3]. Reduced trypanothione is regenerated from its cyclic disulfide form by the NADPH-dependent flavoprotein trypanothione reductase [3]. Reduced tryparedoxin I donates its reducing equivalents to a peroxiredoxin-type protein which, in its reduced form, readily reacts with various hydroperoxides and, in consequence, is classified as a tryparedoxin peroxidase [1,5,7].With a molecular weight of 16 kDa, tryparedoxin I [7] is larger than thioredoxin in vertebrates [8].…”

mentioning

confidence: 99%