Abstract
A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).
Highlights
Fax: 44171 225 0960. 1 The abbreviations used are: GPx, glutathione peroxidases; AcNPV, Autographa californica nuclear polyhedrosis virus; Sf cells, Spodoptera frugiperda cells
Exp ression, Purification, a nd Structural Properties of Recombinant gp29-The full-l en gth cDNA encoding gp29 contai ne d a n N-terminal signal pep tid e t hat had pr eviously been show n to direct t ra ns location of t he pr otein in to micr osoma l membran e pr eparations in vitro [28]. We t he re fore assessed whe t he r gp29 expressed by AcNPV wa s secre te d from t he Sf cells by pul se-ch ase lab elin g wit h [35S1met hionine
Alt hough a si ngle protein of 29 kDa wa s detected in cult ure su pern at ants, cell extracts contained different forms of gp29 ranging in mass from 25 to 32 kDa (Fig. 2A)
Summary
Vol 270, No 31, Issue of August 4, pp. 18313-18318, 1995 Printed in U.S.A. Heterologous Expression and Enzymatic Properties of a Selenium-independent Glutathione Peroxidase from the Parasitic Nematode Brugia pahangi*. A full-length eDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera {rugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). Cytosolic GPxs can reduce HzOz and fatty acid hydroperoxides but not phospholipid hydroperoxides [7], whereas recent data demonstrate that the plasma enzymes show activity against the latter substrates and cholesterol 7ex hydroperoxide [8, 9]. Selenocysteine-independent GPx activity has been reported in the literature, the majority of these cases are due to structurally unrelated glutathione-S-transferases that reduce fatty acid hydroperoxides by an entirely different mechanism. We expressed gp in insect cells via a baculovirus vector in order to examine its putative enzyme activity, and we report here on the properties of this selenium-independent GPx
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