Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains
[EN] The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fl uorescence confocal microscopy. Both the lipids and the enzyme were labeled with specifi c fl uorescent markers. GUV consis...
| Autores: | , , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2011 |
| País: | España |
| Institución: | Universidad del País Vasco |
| Repositorio: | Addi. Archivo Digital para la Docencia y la Investigación |
| OAI Identifier: | oai:addi.ehu.eus:10810/64693 |
| Acceso en línea: | http://hdl.handle.net/10810/64693 |
| Access Level: | acceso abierto |
| Palabra clave: | ceramides cholesterol diacylglycerol fluorescence microscopy lipid rafts membranes/physical chemistry sphingolipids |
| Sumario: | [EN] The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fl uorescence confocal microscopy. Both the lipids and the enzyme were labeled with specifi c fl uorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin,phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5–10 mol% of the enzyme endproduct ceramide and/or diacylglycerol were occasionally added. Morphological examination of the GUV in the presence of enzyme reveals that, although the enzyme diffuses rapidly throughout the observation chamber, detectable enzyme binding appears to be a slow, random process, with new bound-enzyme-containing vesicles appearing for several minutes. Enzyme binding to the vesicles appears to be a cooperative process. After the initial cluster of bound enzyme is detected, further binding and catalytic activity follow rapidly. After the activity has started, the enzyme is not released by repeated washing, suggesting a “scooting” mechanism for the hydrolytic activity. The enzyme preferentially binds the more disordered domains, and, in most cases, the catalytic activity causes the disordering of the other domains. Simultaneously, peanut- or fi gure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate. |
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