Abstract
A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.
Highlights
Aerobic organisms are exposed to oxidative stress in the presence of an excess of reactive oxygen species (ROS) generated either under pathological conditions or with variations in the oxygen tension [1,2,3]
Starting with the cytosolic fraction obtained from D. dorotocephala biomass, it was possible to isolate both disulfide form of glutathione (GSSG) reductase and Trx reductase activities
The sensitivity of the glutathione reductase (GR) activity to auranofin of the two fractions was different, being higher for Pool 1. These results suggest in D. dorotocephala a thioredoxin reductase (TrxR) is expressed as a functional protein
Summary
Aerobic organisms are exposed to oxidative stress in the presence of an excess of reactive oxygen species (ROS) generated either under pathological conditions or with variations in the oxygen tension [1,2,3]. Cells have developed both enzymatic and non-enzymatic protective mechanisms [4,5,6,7,8]. Significant in this sense are the glutathione (GSH) and thioredoxin (Trx) antioxidant dependent systems, which have a broad distribution in the living world [9,10]. Both enzymes are NADPH-dependent homodimeric flavoproteins, and its disulfide reductase activity rely upon a conserved thiol/disulfide redox motif [11,12]
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