A Turbidometric Assay for Phospholipase C and Sphingomyelinase

Torley, Lawrence W.; Silverstrim, Christine; Pickett, Walter C.

doi:10.1006/abio.1994.1517

Cited by 6 publications

(8 citation statements)

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“…Many authors have shown that PLC activity induces vesicles aggregation (Torley et al, 1994;Basáñez et al, 1996Basáñez et al, , 1997. We observed vesicle aggregation with LUVs (data not shown), but not with giant vesicles, due to the low lipid concentration.…”

Section: Vesicle Morphologysupporting

confidence: 46%

“…In addition to performing a phosphate essay, sample turbidity was employed to monitor enzyme activity. An increase in turbidity corresponds to vesicle aggregation induced by the increasing presence of diacylglycerol (Torley et al, 1994;Basáñez et al, 1996Basáñez et al, , 1997. Dynamic light scattering was used to follow the size of the aggregates with time.…”

Section: Enzyme Activitymentioning

confidence: 99%

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Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

Riske

Döbereiner

2003

Biophysical Journal

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We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (alpha-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs.

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Section: Vesicle Morphologysupporting

confidence: 46%

Section: Enzyme Activitymentioning

confidence: 99%

Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

Riske

Döbereiner

2003

Biophysical Journal

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“…Since the SANS measurements show that there is no observable change in liposome morphology and aggregation upon extended incubation with high amounts of PLD, it was necessary to qualify if the PLD was indeed active on membrane-immobilized PC lipids formulated into vesicles. This is nontrivial; many conventional assays for both PLD and PLC measure enzyme activity on fluorescent or radio-labeled free lipids in solution, − while the readout mechanism of liposome-based activity assays relies on release of encapsulated contents or increased aggregation caused by a change in lipid phase behavior. , It has been observed experimentally that incubating PC liposomes with PLC leads to release of encapsulated cargo; however incubation of PC liposomes with PLD leads to stable, asymmetric vesicles . To confirm enzyme activity on intact PC liposomes we used SPR, a highly sensitive technique that quantifies changes in mass of substrate immobilized on a functionalized gold chip, to measure the rate of detachment of PC liposomes from a Biacore L1 chip in the presence of PLD and PLC.…”

Section: Resultsmentioning

confidence: 99%

Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement

et al. 2018

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Understanding the origins of lipid membrane bilayer rearrangement in response to external stimuli is an essential component of cell biology and the bottom-up design of liposomes for biomedical applications. The enzymes phospholipase C and D (PLC and PLD) both cleave the phosphorus–oxygen bonds of phosphate esters in phosphatidylcholine (PC) lipids. The atomic position of this hydrolysis reaction has huge implications for the stability of PC-containing self-assembled structures, such as the cell wall and lipid-based vesicle drug delivery vectors. While PLC converts PC to diacylglycerol (DAG), the interaction of PC with PLD produces phosphatidic acid (PA). Here we present a combination of small-angle scattering data and all-atom molecular dynamics simulations, providing insights into the effects of atomic-scale reorganization on the supramolecular assembly of PC membrane bilayers upon enzyme-mediated incorporation of DAG or PA. We observed that PC liposomes completely disintegrate in the presence of PLC, as conversion of PC to DAG progresses. At lower concentrations, DAG molecules within fluid PC bilayers form hydrogen bonds with backbone carbonyl oxygens in neighboring PC molecules and burrow into the hydrophobic region. This leads initially to membrane thinning followed by a swelling of the lamellar phase with increased DAG. At higher DAG concentrations, localized membrane tension causes a change in lipid phase from lamellar to the hexagonal and micellar cubic phases. Molecular dynamics simulations show that this destabilization is also caused in part by the decreased ability of DAG-containing PC membranes to coordinate sodium ions. Conversely, PLD-treated PC liposomes remain stable up to extremely high conversions to PA. Here, the negatively charged PA headgroup attracts significant amounts of sodium ions from the bulk solution to the membrane surface, leading to a swelling of the coordinated water layer. These findings are a vital step toward a fundamental understanding of the degradation behavior of PC lipid membranes in the presence of these clinically relevant enzymes, and toward the rational design of diagnostic and drug delivery technologies for phospholipase-dysregulation-based diseases.

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“…Bc-SMase activity was measured using a turbidometric assay 35 . Briefly, Bc-SMase (50 ng/ml) was treated at 37 C for 60 min with various compounds that were solubilized in dimethylacetoamide.…”

Section: Smase Activitymentioning

confidence: 99%

Novel inhibitor of bacterial sphingomyelinase, SMY-540, developed based on three-dimensional structure analysis

Oda¹,

Imagawa

Kato³

et al. 2013

Journal of Enzyme Inhibition and Medicinal Chemistry

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A Turbidometric Assay for Phospholipase C and Sphingomyelinase

Cited by 6 publications

References 0 publications

Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

Fate of Liposomes in the Presence of Phospholipase C and D: From Atomic to Supramolecular Lipid Arrangement

Novel inhibitor of bacterial sphingomyelinase, SMY-540, developed based on three-dimensional structure analysis

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