Purification of glutathionylspermidine and trypanothione synthetases from Crithidia fasciculata.
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 fasciculata. 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 N1,N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a kcat of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a kcat 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.
Smith
K
K
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, United Kingdom.
Nadeau
K
K
Bradley
M
M
Walsh
C
C
Fairlamb
A H
AH
D019732:Amide Synthases; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D016831:Crithidia fasciculata / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight
D008025:Ligases; D019732:Amide Synthases; C081536:glutathionylspermidine synthetase; C064407:trypanothione synthetase