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.

Sphingomyelin synthesis in helminths: a minireview.

Sphingolipids are a diverse and ubiquitous group of lipids. They are widely distributed in parasites and a number of novel forms have been described. Sphingolipid synthesis has been investigated in the malarial parasite, cestodes, digeneans and nematodes. Although there are differences in detail, the synthetic pathways involved are similar to those found in mammals.

Isolation and partial characterization of a fatty-acid-binding protein from Ascaris suum reproductive tissue.

A 12-kDa fatty-acid-binding protein was purified to homogeneity from Ascaris suum reproductive tissue as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino-acid-sequence analysis of the protein revealed its identity with the ABA-1 allergen protein isolated from A. suum pseudocoelomic fluid. Fatty-acid binding by the protein from A. suum reproductive tissue was investigated using the Lipidex 1000 assay, which revealed the presence of a single class of fatty-acid-binding sites with an apparent dissociation constant for palmitate of about 0.8 microM.

Characterisation and properties of an intracellular lipid-binding protein from the tapeworm Moniezia expansa.

The tapeworm Moniezia expansa contains an extremely abundant cytoplasmic lipid-binding protein (LBP). It is a small protein consisting of 66 amino acids with a molecular mass of 7943 +/- 1.5 Da. The amino acid sequence has been established by Edman degradation and confirmed by PCR analysis. The Moniezia LBP shows no sequence similarity with any previously described binding protein, but does show similarity with antigen B from Echinococcus glanulosus and Echinococcus multilocularis and with Taenia crassiceps antigen. The predicted structure for Moniezia LBP shows four helices and a putative tyrosine kinase site on the loop between helix 1 and 2. Each of the four helices has a well defined hydrophobic face. Studies with fluorescent probes suggest a single hydrophobic binding site. Results indicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand binding, and calculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic environment.

Methionine and cysteine metabolism in Fasciola hepatica.

Radiolabel from the methyl groups of serine and methyltetrahydrofolate was readily incorporated into methionine in adult Fasciola hepatica, and a substantial proportion of the label from [35S]methionine appeared in cysteine. The data suggest that methionine synthesis is via methyltetrahydrofolate-homocysteine methyltransferase and that there is cysteine synthesis from methionine. Cystathionine-beta-synthase and gamma-cystathionase activities were demonstrated in homogenates.

Forskolin as an activator of adenylate cyclase complex of differentiating erythroid bone-marrow cells.

The study concerns the manner in which forskolin activates the adenylate cyclase system of differentiating rabbit bone-marrow erythroid cells. The results presented show that forskolin can stimulate the basal activity of adenylate cyclase in the absence of guanine nucleotides in an in vitro assay containing plasma membranes derived from both dividing and non-dividing cells. In the presence of guanine nucleotide the activation of adenylate cyclase by forskolin is increased, but the effect is not additive and is abolished by the beta-thio analogue of GDP. Addition of forskolin to cell cultures causes a transient increase in the activity of adenylate cyclase, which is maximal by 30 minutes and disappears within 24 hours. The conclusion is made that the effect of forskolin on adenylate cyclase complex of differentiating rabbit bone-marrow erythroblasts is similar to the effect of erythropoietin (Bonanou-Tzedaki et al., 1986) and is transdusing via stimulatory guanine nucleotide-regulatory protein.