Purification of glutathionylspermidine and trypanothione synthetases from crithidia fasciculata

Abstract

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione-glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase-have been identified and purified individually from Crithidia fusciculutu. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N ' , NE-bis(glutathiony1)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kc,, of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kc,, of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions (0.0-0.15 M NaCl), consistent with a functional complex in vivo.Keywords: amide-forming synthetases; glutathione metabolism; glutathionylspermidine synthetase; polyamine metabolism; trypanothione synthetase The polyamines (putrescine, spermidine, and spermine) and GSH are found in millimolar concentrations in most biological systems. They are thought to be involved in cell growth and differentiation and regulation of important enzymatic processes. GSH plays an important role in the management of oxidative stress and in the maintenance and the regulation of intracellular thiol/disulfide redox balance. Polyamines are required for optimal growth in many cell types, although their precise regulatory functions remain obscure (Tabor & Tabor, 1984). The majority of intracellular GSH of the trypanosomatid parasites,

Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase from Crithidia fasciculata

“…Amino Acid Sequencing-As already observed by Smith et al (1), N-terminal amino acid sequencing proved unsuccessful, obviously due to an N-terminal blocking group. After proteolytic cleavage with endoproteinase Lys-C, however, a total of 11 peptides could be recovered from HPLC in a quality to allow sequencing.…”

“…The assay for trypanothione synthetase (TS) was carried out as described by Smith et al (1). Aliquots were taken after 20 min.…”

“…The first enzyme of TSH synthesis, GspS, has been isolated once in trace amounts from C. fasciculata (0.5 mg from 500 g, wet cell mass) and preliminarily characterized in terms of the apparent M r , kinetic parameters, and substrate specificity (1). An enzyme catalyzing the analogous reaction in E. coli has recently been cloned.…”

Trypanothione Synthesis in Crithidia Revisited

“…This model did not consider the presence of amidase activity that re-converts the GSH spermidine conjugates back to GSH and spermidine nor a distinct GspS. B, scheme of Smith et al (17). It proposes a stepwise biosynthesis of trypanothione catalyzed by two distinct enzymatic entities, Cf-GspS and Cf-TryS.…”

Identification and Biosynthesis of N1,N9‐Bis(Glutathionyl)Aminopropylcadaverine (Homotrypanothione) in Trypanosoma Cruzi

“…Attempts were made to synthesis glutathionylaminopropylcadaverine enzymically using glutathionylspermidine synthetase and trypanothione synthetase from c. fasciculata [31]. Following incubation with a partially purified preparation (approximately 16-fold) containing both enzymes with aminopropylcadaverine in place of spermidine, the mixture was derivatised with monobromobimane and analysed by HPLC.…”