Marine organisms have been less explored than mammals and microorganisms even though there are higher probabilities to find enzymes that have different specifics towards non-natural substrates in those environments. The sea anemone Condylactis gigantean is a representative of this family. Recent studies about marine coelenterates venom have demonstrated affinity to that from snakes, based on the pharmacological properties they possess, and the place where this enzymatic activity has been described for some species. In spite of their wide distribution in nature, most of these enzymes have been isolated from snakes, bees and scorpions venoms, as well as from pancreas tissues of mammals and membranes for example, those of mitochondria from hepatic tissues. These enzymes are widely spread in nature and their functional versatility and usefulness have increasingly conditioned their use in Specialized and Applied Biochemistry as well as biotechnological, pharmaceutical, and general industries such as food industry, where a highly purified enzyme is not always needed. It also reveals maximum activity when the substrate is organized in large molecular aggregations such as micelles. These enzymes yield a lyso derivative (lysophospholipid) and a free-fatty acid molecule, which is able to hydrolyze both natural and synthetic phospholipids regardless of the nature of the fatty acids in the molecule. Phospholipases A 2 (PLA 2) are esterolytic enzymes with high substrate and bond specificity for the hydrolysis of the sn-2 position of phosphoacylglycerols. Keywords: affinity, phospholipase A 2, Condylactis gigantea, sea anemone, protein purification.
These components are produced with molecular weights between 1800, and at least one component possessing phospholipase A 2 activity. By means of the above-mentioned support, purification of three protein-based components can be carried out. The phospholipase A 2 activity was corroborated by using qualitative TLC and a fluorogenic substrate. PLA 2 purification from the sea anemone Condylactis gigantean is conducted through a chromatographic affinity support MANA-Sepharose CL 4B with covalently immobilized phosphatidylcholine egg. This research study aims to test a two-step purification procedure for phospholipases A 2 (PLA 2) to filter enzymes for further characterization. Marine coelenterate venom is composed of complex mixtures of several substances, mainly of proteins, for which phospholipase A 2 activity has been described. Palabras clave: cromatografía de afinidad, fosfolipasa A 2, anémona marina, Condylactis gigantea, purificación de proteínas. Dicho soporte cromatográfico permite que en un protocolo de purificación de solamente dos pasos se obtengan tres componentes proteicos de pesos moleculares entre 18000 y 14000, con al menos un componente que posee actividad fosfolipásica A 2. Se muestra la purificación de FLA 2 en un soporte cromatográfico de afinidad con fosfatidilcolina de huevo inmovilizada covalentemente para la purificación de fosfolipasas A 2, a los que se les comprobó cualitativamente la actividad fosfolipásica A 2 por cromatografía en placa (TLC) utilizando sustrato marcado con fluorescencia.
El objetivo de la presente investigación fue purificar las fosfolipasas A 2 (FLA 2) procedentes de la anémona marina Condylactis gigantea a partir de un protocolo de dos etapas para su posterior caracterización. IFacultad de Ciencias Naturales, Universidad de Oriente, Santiago de Cuba, Cuba, de Estudio de Proteínas (CEP), Facultad de Biología, Universidad de La Habana, Ciudad Habana, Cuba, de Catálisis y Petroleoquímica, CSIC, Campus UAM, Madrid, España, de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, Fiocruz Rondônia e Departamento de Medicina, Universidade Federal de Rondônia, Porto Velho-RO, Brazil, veneno de los celenterados marinos constituyen mezclas complejas de varios componentes, muchos de naturaleza proteica, y para los cuales se ha descrito la actividad fosfolipásica A 2. Dolores Lázara Romero-Del-Sol I I, José Manuel Guisán-Seijas I I I, Joaquín Díaz-Brito II Dr.C. Protocolo de purificación en dos etapas de fosfolipasas A 2 a partir de la anémona marina Condylactis giganteaĪ Two-Step Purification Procedure of Phospholipases A 2 from the Sea Anemone Condylactis gigantea
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