Purification and characterization of trypanothione reductase from Crithidia fasciculata, a new member of the family of disulfide-containing flavoprotein reductases
- 1 June 1986
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 25 (12) , 3519-3526
- https://doi.org/10.1021/bi00360a007
Abstract
Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 .mu.M, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53,800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a .lambda.max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5''-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (.+-. 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (.+-. 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed. The active-site peptide of trypanothione reductase was isolated by alkylation of NADPH-reduced enzyme with iodo[1-14C]acetamide followed by trypsinization of the protein and HPLC purification of the labeled peptide. The labeled peptide was sequenced 24 residues and found to contain 2 cysteine moieties. A comparison of the amino acid sequence of the purified peptide with that of the active-site peptide of glutathione reductase from human erythrocytes revealed homology through 14 residues. However, these two enzymes showed mutually exclusive substrate specificities for their respective disulfide-containing substrates. Thus, trypanothione reductase is a new member of the family of disulfide-containing flavoproteins that includes glutathione reductase, lipoamide dehydrogenase, and mercuric reductase.This publication has 18 references indexed in Scilit:
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