The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) uniquely contains three cGMP binding sites, but also has a ‘degenerate’ fourth site. The role of each cGMP-binding site in PfPKG activation remains unknown. We have analysed the effect of mutation of each cGMP-binding site (individually and in combination) on PfPKG activation in vitro . The most striking result was that mutation of cGMP site 3 resulted in a 10–49-fold increase in the K a (cGMP) value and a 45–55% decrease in maximal activity compared with wild-type. Mutations involving only cGMP-binding sites 1 and 2 had less effect on both the K a (cGMP) values and the maximal activities. These results suggest that, although all three cGMP-binding sites are involved in PfPKG activation, cGMP-binding site 3 has the greatest influence on activation. A mutation in the fourth, degenerate cGMP-binding site decreased PfPKG maximal activity by 40%, but did not change the K a (cGMP) value for the PfPKG mutant, suggesting that this site does not bind cGMP, but is required for full activation of PfPKG. The distinct activation properties of PfPKG from mammalian isoforms may be exploitable in the design of a parasite-specific inhibitor and development of a novel anti-malarial drug.

Until recently, it had been thought that trypanosomes lack glutathione peroxidase activity. Here we report the subcellular localization and biochemical properties of a second glutathione-dependent peroxidase from Trypanosoma cruzi (TcGPXII). TcGPXII is a single-copy gene which encodes a 16kDa protein that appears to be specifically dependent on glutathione as the source of reducing equivalents. Recombinant TcGPXII was purified and shown to have peroxidase activity towards a narrow substrate range, restricted to hydroperoxides of fatty acids and phospholipids. Analysis of the pathway revealed that TcGPXII activity could be readily saturated by glutathione and that the peroxidase functioned by a Ping Pong mechanism. Enzyme reduction was shown to be the rate-limiting step in this pathway. Using immunofluorescence, TcGPXII was shown to co-localize with a homologue of immunoglobulin heavy-chain binding protein (BiP), a protein restricted to the endoplasmic reticulum and Golgi. As the smooth endoplasmic reticulum is the site of phospholipid and fatty acid biosynthesis, this suggests that TcGPXII may play a specific role in the T. cruzi oxidative defence system by protecting newly synthesized lipids from peroxidation.

GSH-dependent prostaglandin D 2 synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli , and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H 2 to PGD 2 . Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K m values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K m for GSH (8mM versus 0.3mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/ l -dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and d -dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4×10 6 M −1 ·s −1 ) was comparable with that of human MIF, while that of p -hydroxyphenylpyruvate (9.1×10 4 M −1 ·s −1 ) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of l -dopachrome compared with non-esterified l -dopachrome (>87000-fold) and a high k cat (≈ 4×10 4 s −1 ) . The crystal structure, determined to 1.65 Å (1 Å = 0.1nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p -hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5ng/ml–5pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6ng/ml (55ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi . We have isolated a single-copy gene from T. cruzi with the potential to encode an 18kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli . The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide> cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.

Macrophage-migration-inhibition factor (MIF) is an essential stimulator of mammalian T-lymphocyte-dependent adaptive immunity, hence MIF orthologues might be expressed by infectious organisms as an immunosubversive stratagem. Since MIF actively catalyses the tautomerization of the methyl ester of l -dopachrome (using dopachrome tautomerase), the occurrence of MIF orthologues in several parasitic helminths was investigated by assaying and characterizing such activity. Evidence of MIF orthologues (dopachrome tautomerase) was found in the soluble fraction of the nematodes Trichinella spiralis (stage 4 larvae) and Trichuris muris (adults), and the filarial nematode Brugia pahangi (adults). The MIF orthologues of Tr. muris (TmMIF) and B. pahangi (BpMIF) were purified to homogeneity using phenyl-agarose chromatography, that of T. spiralis (TsMIF) required a further step: cation-exchange FPLC. Retention time on reverse-phase HPLC and M r on SDS/PAGE of the nematode MIFs were similar to those of human MIF. N-terminal sequences (19 residues) of TsMIF and TmMIF showed 47 and 36% identity, respectively, with human MIF. The N-terminal sequence of BpMIF (14 residues) was identical to that of an MIF orthologue in the genome of B. malayi (Swiss-Prot, P91850) and showed 43% identity to either human or TsMIF. TsMIF had 10-fold higher dopachrome tautomerase activity than MIF from the other sources. The enzyme activities of TsMIF, BpMIF and TmMIF were less sensitive to inhibition by haematin ( I 50 : > 15 µ M, > 15 µ M and 2.6 µ M, respectively) than that of human MIF ( I 50 0.2 µ M). Significant dopachrome tautomerase or phenyl-agarose-purifiable MIF-like protein was not detected in the soluble fraction of the nematodes Heligmosomoides polygyrus and Nippostrongylus brasiliensis , the cestode Hymenolepis diminuta , or the trematodes Schistosoma mansoni , S. japonicum and S. haematobium , or the free-living nematode, Caenorhabditis elegans , which does contain an MIF-related gene.

The Expressed Sequence Tag database has been screened for cDNA clones encoding prostaglandin D 2 synthases (PGDSs) by using a BLAST search with the N-terminal amino acid sequence of rat GSH-dependent PGDS, a class Sigma glutathione S-transferase (GST). This resulted in the identification of a cDNA from chicken spleen containing an insert of approx. 950 bp that encodes a protein of 199 amino acid residues with a predicted molecular mass of 22732 Da. The deduced primary structure of the chicken protein was not only found to possess 70% sequence identity with rat PGDS but it also demonstrated more than 35% identity with class Sigma GSTs from a range of invertebrates. The open reading frame of the chicken cDNA was expressed in Escherichia coli and the purified protein was found to display high PGDS activity. It also catalysed the conjugation of glutathione with a wide range of aryl halides, organic isothiocyanates and α,β-unsaturated carbonyls, and exhibited glutathione peroxidase activity towards cumene hydroperoxide. Like other GSTs, chicken PGDS was found to be inhibited by non-substrate ligands such as Cibacron Blue, haematin and organotin compounds. Western blotting experiments showed that among the organs studied, the expression of PGDS in the female chicken is highest in liver, kidney and intestine, with only small amounts of the enzyme being found in chicken spleen; in contrast, the rat has highest levels of PGDS in the spleen. Collectively, these results show that the structure and function, but not the expression, of the GSH-requiring PGDS is conserved between chicken and rat. The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, GSDB and DDBJ Nucleotide Sequence Databases under the accession number AJ006405.

Macrophage-migration-inhibitory factor (MIF) is retained by S -hexylglutathione–agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Åman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med. 2 , 143–149]. The l -dopachrome methyl ester tautomerase activity of human liver MIF was not inhibited by a variety of glutathione S-conjugates, eicosanoids or glucocorticoids but was very sensitive to inhibition by haematin (IC 50 100–200 nM). The inhibition was non-competitive and showed positive co-operativity ( h = 5.8). Similar sensitivity to haematin was obtained with purified recombinant human MIF. The sensitivity of MIF to haematin is approx. 1000-fold greater than for any previously described ligands, and is within its physiological range. Therefore the interaction is likely to be important in modulating the function of MIF in the initiation of immune responses.

Comparison of partial primary sequences of sigma-class glutathione S-transferases (GSH) of parasitic helminths and a GSH-dependent prostaglandin (PG)-H D -isomerase of rat immune accessory cells suggested that some of the helminth enzymes may also be involved in PG biosynthesis [Meyer and Thomas (1995) Biochem. J. 311 , 739-742]. A soluble GSH transferase of the parasitic nematode Ascaridia galli has now been purified which shows high activity and specificity in the GSH-dependent isomerization of PGH to PGE, comparable to that of the rat spleen enzyme in its isomerization of PGH to PGD, and similarly stimulates the activity of prostaglandin H synthase. The enzyme subunit is structurally related to the rat spleen enzyme and sigma-class GSH transferases of helminths according to the partial primary sequence. The data support the hypothesis that some sigma-class GSH transferases of helminth parasites are involved in PG biosynthesis which, in the case of PGE, is likely to be associated with the subversion or suppression of host immunity. A PG-H E-isomerase of comparable specificity and activity has not previously been isolated.