RESUMO
Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10(-3)U/mg of protein). In this work, a mixture StI-StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI-StII was relatively high (1.85U/mg) and dependent of Ca(2+). The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40mM Ca(2+) at pH 8.0. This Ca(2+) concentration did not exert any effect on binding of StI-StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.
Assuntos
Venenos de Cnidários/metabolismo , Proteínas Hemolisinas/metabolismo , Fosfolipases A2/metabolismo , Anêmonas-do-Mar/química , Anêmonas-do-Mar/enzimologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Cromatografia de Afinidade , Venenos de Cnidários/química , Venenos de Cnidários/isolamento & purificação , Proteínas Hemolisinas/química , Proteínas Hemolisinas/isolamento & purificação , Dados de Sequência Molecular , Compostos Orgânicos/química , Compostos Orgânicos/isolamento & purificação , Compostos Orgânicos/metabolismo , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Alinhamento de SequênciaRESUMO
This work aimed at the isolation and structural/functional characterization of a phospholipase A(2) (CgPLA(2)) from the extract of the anemone Condylactis gigantea. CgPLA(2) was isolated with a high purity level through three chromatographic steps, showing pI 8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA(2) showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca(2+)-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA(2) with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA(2) revealed 84%, 61%, 43% and 42% similarity to the PLA(2)s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively.