RESUMO
An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) aspartic proteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 degrees C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general aspartic-proteinase inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea-fan coral) Plexaura homomalla (black sea rod), with increases of 10.2- and 5.9-fold in the specific inhibitory activity respectively. The preliminary K(i) values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II inhibitors from complex mixtures and established the presence of Plm II inhibitors in some marine invertebrates.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Produtos Biológicos/isolamento & purificação , Cromatografia de Afinidade/métodos , Inibidores Enzimáticos/isolamento & purificação , Animais , Antozoários/química , Antimaláricos/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários , Reprodutibilidade dos Testes , Xestospongia/químicaRESUMO
Sticholysin II (St II) is a cytolysin produced by the sea anemone Stichodactyla helianthus, characterized by forming oligomeric pores in natural and artificial membranes. In the present work the influence of the membrane lipidic components sphingomyelin (SM) and cholesterol (Cho) on binding and functional activity of St II, was evaluated using ELISA, lipid monolayers and liposomes. The aim of this work was to establish the promoting role of Cho and SM, both in St II binding and pore formation efficiency. In general the association (evaluated by ELISA and incorporation to phospholipid monolayers) of St II to lipids mixtures was better than to any one of the single components. Regarding the unique role of SM, it was found that, albeit inefficiently, St II binds to phosphatidylcholine (PC):Cho monolayers and liposomes, and is able to form active pores in these bilayers. The results in monolayers and liposomes show that the presence of SM and large amounts of Cho leads to the highest values of critical pressure and rate of association to monolayers, the most favorable interaction with liposomes, and the fastest rate of pore formation, in spite of the rigidity of the layers as suggested by the high generalized polarization (GP) of Laurdan incorporated to liposomes and FTIR data. Taken together, the present results show that the joint presence of SM and Cho, both in binary and ternary (PC containing) mixtures provide conditions particularly suitable for St II binding and function. We suggest that microdomains present in the bilayers could be important for toxin-membrane association.
Assuntos
Colesterol/farmacologia , Venenos de Cnidários/farmacologia , Lipídeos de Membrana/metabolismo , Esfingomielinas/farmacologia , Animais , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática , Lipossomos/metabolismo , Ligação Proteica , Anêmonas-do-MarRESUMO
Recent research suggests that marine organisms may produce compounds with activity against malaria parasites. Of a total of 27 aqueous extracts from different marine species, collected on the northwest Cuban coast, 20 were considered as showing no significant activity against Plasmodium falciparum F32, with minimum inhibitory concentrations (MIC) >500 microg/ml, while seven extracts (MIC < or =500 microg/ml) were selected for further investigation by determining their selectivity indices and in vivo antimalarial activity. Three species of tunicates were chosen, as more than 50% reduction of P. berghei parasitaemia was produced after administration of 250 or 500 mg/kg of their crude extracts into infected mice. The aqueous extracts of Microcosmus goanus, Ascidia sydneiensis and Phallusia nigra were partitioned between water and n-butanol; the organic phases inhibited P. falciparum growth by 50% at concentrations of 17.5 microg/ml, 20.9 microg/ml and 29.4 microg/ml respectively. In general, these results are similar to those of most ethnobotanical surveys. Further chemical studies are being undertaken in order to isolate new antimalarial compounds from these Caribbean tunicates.