Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains.
The binding and early stages of activity of a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa on giant unilamellar vesicles (GUV) have been monitored using fluorescence confocal microscopy. Both the lipids and the enzyme were labeled with specific fluorescent markers. GUV consisted of a mixture of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol in equimolar ratios, to which 5-10 mol% of the enzyme end-product 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 figure-eight-shaped vesicles containing two separate lipid domains become spherical. At a further stage of lipid hydrolysis, lipid aggregates are formed and vesicles disintegrate.
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Maitane
M
Ibarguren
David J
DJ
López
L-Ruth
LR
Montes
Jesús
J
Sot
Adriana I
AI
Vasil
Michael L
ML
Vasil
Félix M
FM
Goñi
Alicia
A
Alonso
D002518:Ceramides / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D004075:Diglycerides / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry*
C011439:1,2-diacylglycerol; D002518:Ceramides; D004075:Diglycerides; D053835:Unilamellar Liposomes; D002784:Cholesterol; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase
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