The purification and properties of glutathione reductase from the cestode Moniezia expansa.
Glutathione reductase has a central role in glutathione metabolism and as such is a potential target for chemotherapy. The aim of the work was to purify and characterise glutathione reductase from the cestode Moniezia expansa and to compare the properties of the helminth enzyme with its mammalian counterpart. The enzyme was purified by a combination of anion exchange and affinity chromatography and further characterized by chromatofocusing and gel electrophoresis. Analysis revealed a single isoenzyme of glutathione reductase in Moniezia expansa, with a pI of 5.8. The enzyme was a homodimer of native molecular weight 114 kDa, subunit weight 63 kDa. Enzyme activity was affected by buffer concentration and the presence of monovalent sodium salts. The pH optimum was 7.4 with NADPH as cofactor and 5 with NADH. The Kma for oxidized glutathione was 76 microM and for NADPH and NADH, 21 and 350 microM respectively. In addition to oxidized glutathione only the mixed disulphide between CoA and glutathione (CoASSG) showed any significant activity as substrate. The cestode enzyme was inhibited by a variety of compounds including arsonic derivatives, 2,4,6 trinitrobenzene sulfonate 1,3-bis (2-chlorethyl)-1-nitrosourea and oxidized glutathione. In conclusion the glutathione reductase of M. expansa resembles the mammalian enzyme in its general physical properties and its substrate and inhibitor profile. However, the parasite enzyme shows an unusually high activity with the mixed disulphide of coenzyme A and glutathione (CoASSG) and appears to be more sensitive to inhibition by sodium ions.
McCallum
M J
MJ
Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, U.K.
Barrett
J
J
D000818:Animals; D002021:Buffers; D002589:Cestoda / Q000201:enzymology*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013379:Substrate Specificity
D002021:Buffers; D005980:Glutathione Reductase