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: | , , , , , , , |
|---|---|
| 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 |
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Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domainsIbarguren, MaitaneLópez Jiménez, DavidMontes Burgos, Lidia RuthSot Sanz, JesúsVasil, Michael L.Vasil, Adriana I.Goñi Urcelay, Félix MaríaAlonso Izquierdo, Aliciaceramidescholesteroldiacylglycerolfluorescence microscopylipid raftsmembranes/physical chemistrysphingolipids[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.Spanish Ministerio de Ciencia e Innovación Grants BFU 2008-01637/BMC and BFU 2007-62062 Basque Government Grant IT 461-07 ETORTEK 07/26 National Institutes of Health Grant HL-062608 Basque Government.Elsevier202420242011info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10810/64693reponame:Addi. Archivo Digital para la Docencia y la Investigacióninstname:Universidad del País VascoIngléshttps://www.sciencedirect.com/science/article/pii/S0022227520408983?via%3Dihubinfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by/4.0/© 2011 by the American Society for Biochemistry and Molecular Biology, Inc. This is an Open Access article under the CC BY licenseoai:addi.ehu.eus:10810/646932026-06-18T09:23:17Z |
| dc.title.none.fl_str_mv |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| title |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| spellingShingle |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains Ibarguren, Maitane ceramides cholesterol diacylglycerol fluorescence microscopy lipid rafts membranes/physical chemistry sphingolipids |
| title_short |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| title_full |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| title_fullStr |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| title_full_unstemmed |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| title_sort |
Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting order-disorder and gel-fluid domains |
| dc.creator.none.fl_str_mv |
Ibarguren, Maitane López Jiménez, David Montes Burgos, Lidia Ruth Sot Sanz, Jesús Vasil, Michael L. Vasil, Adriana I. Goñi Urcelay, Félix María Alonso Izquierdo, Alicia |
| author |
Ibarguren, Maitane |
| author_facet |
Ibarguren, Maitane López Jiménez, David Montes Burgos, Lidia Ruth Sot Sanz, Jesús Vasil, Michael L. Vasil, Adriana I. Goñi Urcelay, Félix María Alonso Izquierdo, Alicia |
| author_role |
author |
| author2 |
López Jiménez, David Montes Burgos, Lidia Ruth Sot Sanz, Jesús Vasil, Michael L. Vasil, Adriana I. Goñi Urcelay, Félix María Alonso Izquierdo, Alicia |
| author2_role |
author author author author author author author |
| dc.subject.none.fl_str_mv |
ceramides cholesterol diacylglycerol fluorescence microscopy lipid rafts membranes/physical chemistry sphingolipids |
| topic |
ceramides cholesterol diacylglycerol fluorescence microscopy lipid rafts membranes/physical chemistry sphingolipids |
| description |
[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. |
| publishDate |
2011 |
| dc.date.none.fl_str_mv |
2011 2024 2024 |
| dc.type.none.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/10810/64693 |
| url |
http://hdl.handle.net/10810/64693 |
| dc.language.none.fl_str_mv |
Inglés |
| language_invalid_str_mv |
Inglés |
| dc.relation.none.fl_str_mv |
https://www.sciencedirect.com/science/article/pii/S0022227520408983?via%3Dihub |
| dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/4.0/ |
| eu_rights_str_mv |
openAccess |
| rights_invalid_str_mv |
http://creativecommons.org/licenses/by/4.0/ |
| dc.format.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Elsevier |
| publisher.none.fl_str_mv |
Elsevier |
| dc.source.none.fl_str_mv |
reponame:Addi. Archivo Digital para la Docencia y la Investigación instname:Universidad del País Vasco |
| instname_str |
Universidad del País Vasco |
| reponame_str |
Addi. Archivo Digital para la Docencia y la Investigación |
| collection |
Addi. Archivo Digital para la Docencia y la Investigación |
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15,300724 |