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R D Walter

Publications and source records attributed to R D Walter.

At least 37 records · Page 2Linked to original sources
PubMed2000

Deletion of the parasite-specific insertions and mutation of the catalytic triad in glutathione reductase from chloroquine-sensitive Plasmodium falciparum 3D7.

The flavoenzyme glutathione reductase (GR; NADPH+glutathione disulphide+H(+)-->NADP(+)+2 glutathione-SH) of Plasmodium falciparum is a promising drug target against tropical malaria. As P. falciparum genes are assumed to be highly polymorphic we have cloned and expressed the GR cDNA of the chloroquine-sensitive strain 3D7. In comparison to the known GR of the chloroquine-resistant K1 strain there are three base exchanges all of them leading to amino acid substitutions (residues 281, 285 and 335). The catalytic efficiency k(cat)/K(m) of the 3D7 enzyme is 5-fold lower than for the K1 enzyme. In contrast, vis-à-vis the drugs carmustine, methylene blue and fluorophenyliso-alloxazine the two enzyme species exhibited identical inhibition kinetics. Two structural motifs which are specific for P. falciparum GR were studied by mutational deletion analysis of 3D7 GR. Loop 126-138 appears to be important for folding and stability of the enzyme, whereas the subdomain 318-350 was found to be involved in FAD-binding. The subdomain has no major influence on the known functions of the catalytic triad Cys-40, Cys-45 and His-485'. Flavin absorption spectroscopy of inactive point mutants showed that Cys-45 forms a thiolate charge transfer complex and Cys-40 is the interchange thiol, which reduces glutathione disulphide. The mutant His-485-->Gln had a normal K(m) for glutathione disulphide reduction but only 0.8% residual catalytic activity when compared with wild-type GR, which confirms its function as an acid/base catalyst. The parasite-specific domains in combination with the reactive catalytic residues appear to be a suitable target matrix for inhibiting GR in vivo.

Amino Acid Sequence↗
PubMed2000

In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase.

The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins. We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region. The predicted molecular mass of the entire transcript is 166 kDa. The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331. The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P. falciparum. SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain. The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P. falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs.

Adenosylmethionine Decarboxylase↗
PubMed2000

Plasmodium falciparum-infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione.

During the erythrocytic cycle, Plasmodium falciparum is highly dependent on an adequate thiol status for its survival. Glutathione reductase as well as de novo synthesis of GSH are responsible for the maintenance of the intracellular GSH level. The first and rate-limiting step of the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). Using L-buthionine-(S, R)-sulphoximine (BSO), a specific inhibitor of the gamma-GCS, we show that the infection with P. falciparum causes drastic changes in the GSH metabolism of red blood cells (RBCs). Infected RBCs lose GSH at a rate 40-fold higher than non-infected RBCs. The de novo synthesis of the tripeptide was found to be essential for parasite survival. GSH depletion by BSO inhibits the development of P. falciparum with an IC(50) of 73 microM. The effect of the drug is abolished by supplementation with GSH or GSH monoethyl ester. Our studies demonstrate that the plasmodicidal effect of the inhibitor BSO does not depend on its specificity towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite GSH in the P. falciparum-infected RBCs. Therefore the depletion of GSH is proposed as a chemotherapeutic strategy for malaria, and gamma-GCS is proposed as a potential drug target.

Animals↗
PubMed2000

Onchocerca volvulus superoxide dismutase genes: identification of functional promoters for pre-mRNA transcripts which undergo trans-splicing.

The genes encoding three forms of superoxide dismutase, the cytosolic and extracellular CuZn superoxide dismutases and the mitochondrial Mn superoxide dismutase, were isolated from an Onchocerca volvulus lambda fix II genomic library. Genomic Southern blot analyses indicate single-copy genes in the O. volvulus genome. The O. volvulus cytosolic and extracellular CuZnSOD genes (Ov-sod-1 and Ov-sod-2) are separated by 0.8 kb of sequence and are convergently transcribed. Since the transcripts from all three sod genes are trans-spliced, the transcription start point of each gene was determined in a heterologous system that lacks trans-splicing machinery by in vitro transcription using Drosophila embryo nuclear extracts, followed by primer extension experiments. The ability of the 5' flanking region of the genes encoding the three Ov-SODs to promote transcription was further examined in transient transfections of Chinese hamster ovary cells. In firefly luciferase reporter assays, the Ov-sod-1 and -2 and the MnSOD (Ov-sod-3) gene promoters showed minimal, strong, and moderate levels of activity in these cells, respectively. Both Ov-sod-2 and -3 gene promoter regions showed an initial increase in activity in response to 5' deletions. The results from the in vitro transcription experiments and the luciferase reporter assays were consistent and suggest the presence of Inr-like elements in the promoter regions of the Ov-sod genes.

Animals↗
PubMed2000

Identification of a stress-responsive Onchocerca volvulus glutathione S-transferase (Ov-GST-3) by RT-PCR differential display.

The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.

Amino Acid Sequence↗
PubMed2000

The gamma-glutamylcysteine synthetase of Onchocerca volvulus.

The tripeptide glutathione (GSH) plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. The intracellular GSH level depends on glutathione reductase as well as on GSH synthesis. The first and rate limiting step in the synthetic pathway is catalysed by gamma-glutamylcysteine synthetase (gamma-GCS). The gamma-GCS was partially purified from the filarial parasite Onchocerca volvoulus and preliminary steady state kinetics were performed. The Ki-value for L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of gamma-GCS, was determined to be 0.13 microM, which is 54-fold lower than the Ki-value for the mammalian enzyme. Filarial gamma-GCS was also inhibited by cystamine with a Ki-value of 3.9 microM compared with 22.2 microM determined for the rat enzyme. Further, the cDNA and the gene of the O. volvulus gamma-GCS were cloned and sequenced. The gene of 5762 bp is composed of 14 exons and 13 introns. Southern blot analysis indicates that the gamma-GCS gene is present as a single-copy gene. In accordance with Northern blot analysis, the entire cDNA sequence encompasses 2377 bp. At its 5' end a nematode-specific spliced leader 130 bp upstream of the first in frame methionine was identified. The cDNA encodes a polypeptide of 652 amino acids with 50 and 69% sequence identity to the human and the Caenorhabditis elegans counterparts, respectively. The filarial gamma-GCS is proposed as a potential drug target.

Amino Acid Sequence↗
PubMed1999

The putative gamma-glutamylcysteine synthetase from Plasmodium falciparum contains large insertions and a variable tandem repeat.

The tripeptide glutathione plays a pivotal role in the maintenance of the thiol redox state of the cell and for the detoxification of reactive oxygen species. Glutathione is synthesized in two consecutive reactions by y-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase, respectively. The former enzyme represents the rate limiting step of the synthetic pathway. We have cloned the cDNA and gene of a putative gamma-GCS from Plasmodium falciparum. The contiguous cDNA sequences obtained from various cDNA libraries of P. falciparum K1 and 3D7 encompass 4206 bp or 4038 bp and encode polypeptides of 1119 and 1063 amino acids, respectively. The deduced amino acid sequences show four regions of homology (identity: 31.3-43.9%) to human and Trypanosoma brucei gamma-GCS. These regions are interrupted by three large insertions between 94 and 239 amino acids. Within the first insert a variable repetitive motif was identified, which is responsible for the differing sizes of the sequences. We have analysed this phenomenon in five additional P. falciparum strains and found a high degree of variability in the number of the repeated octamer (Y/C)S(N/D)LQQ(Q/R). Therefore the predicted molecular mass of the proteins from different P. falciparum strains ranges from 124.4 to 133.2 kDa, which is almost twice that of the catalytic subunit of the human host enzyme. Isolation of three genomic clones revealed that the gene does not contain introns. P. falciparum gamma-GCS transcription peaks in trophozoites (24-30 h) suggesting that the antioxidant glutathione is predominantly produced at a time where hemoglobin degradation and the simultaneous formation of reactive oxygen species is maximal.

Amino Acid Sequence↗
PubMed1999

Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family.

A fatty acid-binding protein from the nematode Ascaridia galli was characterized. The gene was isolated and recombinantly expressed in Escherichia coli. According to the deduced amino acid sequence A. galli fatty acid-binding protein (AgFABP) belongs to the family of nematode polyprotein allergens, as shown by Western blotting and PCR analysis with genomic DNA and cDNA. Both native and recombinant proteins bind fatty acids and retinoids with high affinity. The fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1-sulfonyl)amino] undecanoic acid (DAUDA) shows substantial changes in its emission spectrum when bound to AgFABP; this binding is reversed by fatty acids such as oleate. Moreover, changes of the intrinsic fluorescence of retinol and retinoic acid confirm retinoid binding activity of AgFABP. Fluorescence titration experiments with DAUDA indicate stoichiometric binding to a single binding site per monomer unit with affinities (Kd) of 1.6 and 1.8 x 10(-7) m for native and the recombinant protein, respectively. The apparent binding affinities of the nonfluorescent ligands were calculated in displacement experiments with DAUDA and values in the same range were obtained for myristic, palmitic, oleic, linoleic, arachidonic and retinoic acid. Additionally, the binding affinity of AgFABP for oleate and palmitate was determined by direct and indirect radiochemical analysis and the values obtained were similar to those from the fluorescent experiments. Both proteins show a preference for the binding of long-chain saturated and unsaturated fatty acids, but not for short chain (C3-C12) and branched fatty acids, cholesterol and tryptophan.

Allergens↗
PubMed1998

Molecular and biochemical characterization of S-adenosylmethionine decarboxylase from the free-living nematode Caenorhabditis elegans.

S-Adenosylmethionine decarboxylase (SAMDC) is a major regulatory enzyme in the polyamine biosynthesis and is considered a potentially important drug target for the chemotherapy of proliferative and parasitic diseases. To study regulatory mechanisms which are involved in the expression of SAMDC of the free-living nematode Caenorhabditis elegans, we have isolated the SAMDC gene and cDNA. Genomic Southern-blot analysis suggests that the C. elegans SAMDC is encoded by a single-copy gene which spans 3.9 kb and consists of six exons and five introns. The first two introns are located in the 5'-untranslated region (UTR). Analyses of the 5'-flanking region of the gene revealed several consensus sequences for the binding of different transcription factors such as CBP, AP2, cMyb, VPE2 and others. The C. elegans SAMDC mRNA possesses an open reading frame (ORF) which encodes a polypeptide of 368 amino acids, corresponding to a SAMDC proenzyme with a calculated molecular mass of 42141 Da. The active form of the C. elegans SAMDC is a heterotetramer, consisting of two subunits of 32 and 10 kDa derived from cleavage of the pro-enzyme. The SAMDC mRNA has an unusually long 5'-UTR of 477 nucleotides. This region has a small ORF which could encode a putative peptide of 17 residues. Moreover, the C. elegans SAMDC mRNA is trans-spliced with the 22 nucleotides spliced leader sequence at the 5'-end.

Adenosylmethionine Decarboxylase↗
PubMed1998

MGBG analogues as potent inhibitors of S-adenosylmethionine decarboxylase of Onchocerca volvulus.

Polyamines are essential for cell growth and differentiation and therefore, S-adenosylmethionine decarboxylase (SAMDC), a key regulatory enzyme of the polyamine biosynthesis, is considered as a potentially important target for chemotherapy of filarial infections. Recombinant Onchocerca volvulus SAMDC was expressed in Escherichia coli and characterised. The enzyme activity was found to be stimulated 15-fold by addition of 1 mM putrescine. The Km-value for S-adenosylmethionine was determined to be 36 microM. Furthermore, the efficiencies of SAMDC inhibitors were analysed: Berenil inhibits the enzyme activity competitively with a Ki-value of 0.1 microM. MDL 73811 acts as an irreversible inhibitor with a Ki-value of 1.4 microM. Recently synthesised aromatic methylglyoxal bis(guanylhydrazone) analogues demonstrated high efficacy as inhibitors of the SAMDCs. Some of these analogues exhibited Ki-values of 5 and 14 nM for the Onchocerca enzyme, a result which shows an up to 100-fold increase in specificity compared to the value of 0.47 microM for methylglyoxal bis(guanylhydrazone). These inhibitors might have potential as drug candidates against filarial worms.

Adenosylmethionine Decarboxylase↗
PubMed1998

Gene structure, activity and localization of a catalase from intracellular bacteria in Onchocerca volvulus.

Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O. volvulus adult lambda zapII cDNA library. Analysis of their nucleotide and encoded amino acid sequences revealed that they derive from intracellular bacteria, rather than the O. volvulus nuclear genome. The endobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene. The endobacterial CAT gene encodes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E. coli expression system. The purified 54 kDa protein has CAT activity over a broad pH range, with a specific activity of 103,000 +/- 3000 U mg(-1). The optical spectrum of the endobacterial CAT shows that it is a ferric haem-containing protein with a Soret band at 405 nm. To investigate the phylogeny of the intracellular bacterium in O. volvulus, a segment of the 16S rRNA gene was amplified from total genomic DNA by a polymerase chain reaction using universal eubacterial primers. A phylogenetic analysis of the O. volvulus-derived 16S rRNA sequence revealed that the endobacterium belongs to a distinct Wolbachia clade of the order Rickettsiales. Onchocercomata and biopsies containing different onchocercal species were immunohistochemically stained using polyclonal antibodies raised against the recombinant endobacterial CAT. CAT was detected in the endobacteria in the hypodermis of adult male and female O. volvulus, O. ochengi, O. gibsoni and O. fasciata. The endobacterial enzyme was also detected in onchocercal oocytes and all embryonic stages including intrauterine microfilariae as well as skin microfilariae. O. volvulus thus harbours Wolbachia-like endosymbionts which are transovarially transmitted and show particular affinity for the hypodermal tissues of the lateral chords.

Amino Acid Sequence↗
PubMed1998

Primary sequence and activity analyses of a catalase from Ascaris suum.

A complete cDNA encoding the catalase (EC 1.11.1.6) has been isolated from the parasitic nematode Ascaris suum (AsCAT). The active-site residues, the residues involved in ligand interaction, and NADPH-binding residues of the bovine liver catalase-type enzyme are highly conserved in the AsCAT predicted amino acid sequence. To confirm that the AsCAT cDNA encodes a functional enzyme, active recombinant protein (rAsCAT) was produced in a procaryotic expression system. The subunit molecular mass of the purified recombinant protein (rAsCAT) was determined to be approximately 60 kDa. According to gel filtration, the molecular mass of the active enzyme is 240 kDa, indicating that the catalase subunits form a homotetramer in solution. The optical spectrum of rAsCAT shows a typical ferric haem spectrum with a Soret band at 407 nm. Fluorescence spectroscopy demonstrates that rAsCAT binds NADPH. rAsCAT has catalase activity with hydrogen peroxide over a broad pH range, with a specific activity of 37,800 U mg-1. In addition to its catalase activity, rAsCAT displays peroxidase activity using the substrates t-butyl hydroperoxide and o-dianisidine. The haem ligands NaN3 and KCN caused a 50% inhibition of catalase activity at 9 and 19 microM, respectively. In the presence of a H2O2-generating system, catalase activity of rAsCAT was inhibited by 3-aminotriazole, phenolic compounds, and drugs.

Amino Acid Sequence↗
PubMed1998

The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.-W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584-29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C-terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant P/TrxRC535AC540A and the deletion mutant PfTrxRdelta9 (C-terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5'-dithiobis (2-nitrobenzoate). These data imply that the C-terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.

Animals↗
PubMed1998

Identification of stress-responsive genes in Caenorhabditis elegans using RT-PCR differential display.

In order to identify genes that are differentially expressed as a consequence of oxidative stress due to paraquat we used the differential display technique to compare mRNA expression patterns in Caenorhabditis elegans . A C.elegans mixed stage worm population and a homogeneous larval population were treated with 100 mM paraquat, in parallel with controls. Induction of four cDNA fragments, designated L-1, M-47, M-96 and M-132, was confirmed by Northern blot analysis with RNA from stressed and unstressed worm populations. A 40-fold increase in the steady-state mRNA level in the larval population was observed for the L-1/M-47 gene, which encodes the detoxification enzyme glutathione S-transferase. A potential stress-responsive transcription factor (M-132) with C2H2-type zinc finger motifs and an N-terminal leucine zipper domain was identified. The M-96 gene encodes a novel stress-responsive protein. Since paraquat is known to generate superoxide radicals in vivo , the response of the C.elegans superoxide dismutase (SOD) genes to paraquat was also investigated in this study. The steady-state mRNA levels of the manganese-type and the copper/zinc-type SODs increased 2-fold in the larval population in response to paraquat, whereas mixed stage populations did not show any apparent increase in the levels of these SOD mRNAs.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell. Recent studies revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases. The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. The putative essential amino acids at the catalytic center of large thioredoxin reductase from P. falciparum were determined by using site-directed mutagenesis techniques. To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues. Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine. All mutants were expressed in the E. coli system and characterized. Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity. These results clearly identify Cys88 and Cys93 as the active site thiols of large thioredoxin reductase. The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity. These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase. Spectral analysis of wild-type P. falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase. This spectral feature, recently also reported for the human placenta protein (Arscott, L. D., Gromer, S., Schirmer, R. H., Becker K., and Williams, C. H., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3621-3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to small E. coli thioredoxin reductase.

Animals↗
PubMed1997

Molecular characterization and expression of Onchocerca volvulus glutathione reductase.

Glutathione metabolism represents a potential target for anti-parasite drug design. The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus. The O. volvulus GR (OvGR) gene was cloned and sequenced. The gene is composed of 13 exons and 12 introns and spans 4065 bp. The first intron is located within the 5'-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon. Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene. Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed. The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5' end, 236 nucleotides upstream of the first in-frame methionine. The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR). A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR. However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR). The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 microM) over NADH (Km 108 microM). The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.

Amino Acid Sequence↗
PubMed1997

Structural and functional analysis of a glutathione S-transferase from Ascaris suum.

A recombinant glutathione S-transferase (GST) (EC 2.5.1.18) from the parasitic nematode Ascaris suum (AsGST1) displays specific activity with a variety of model substrates and secondary products of lipid peroxidation. The AsGST1 interacts with a range of model inhibitors, haematin-related compounds, bile acids and anthelminthics. The reported variations in biochemical activity correlate with structural differences observed by homology modelling. Here, differences in the topography of the proposed substrate binding site between the AsGST1 and the host GSTs were identified. A rabbit polyclonal antiserum was raised against the glutathione-binding proteins of A. suum and specific antibodies against AsGST1 were affinity-purified using the recombinant protein. These antibodies were used to localize the AsGST1 in adult worms by immunohistochemical staining. The strongest immunostaining for AsGST1 was localized in the intestine in all worms examined. This suggests that the enzyme may be responsible for the metabolism of materials that are incorporated from the environment, as well as for molecules that are excreted or secreted from the parasite to the environment. It also demonstrates the accessibility of the enzyme to an inhibitor or blocking antibody. In addition, the structure and sequence of the gene encoding AsGST1 have been determined. Southern-blot analyses of the AsGST1 gene suggests that it is a single-copy gene. The nucleotide sequence analysis revealed that the gene is composed of four exons and three introns, and potential regulatory elements were identified in the 5' flanking sequence.

Amino Acid Sequence↗