Cobra venom phospholipase A2: A review of its action toward lipid/water interfaces

Dennis, Edward A.; Darke, Paul L.; Deems, Raymond A.; Kensil, Charlotte R.; Plückthun, Andreas

doi:10.1007/bf02354830

Cited by 49 publications

(27 citation statements)

References 33 publications

(23 reference statements)

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“…Harvat et al (13) using 2-acyl-lyso-PE generated by the Rhizopus arrhizus lipase determined a K d value for this E. coli homolog 5ϫ lower than our result determined with the LplT-Kp protein and 1-acyl isomer. It may be difficult to determine the stereoselectivity of LplT because compelling evidence from different groups has shown that 2-acyl-lysophospholipids are extremely unstable in biological solution and are quickly converted to the 1-acyl-2-lyso form by a spontaneous intramolecular acyl migration, yielding a mixture mainly containing 1-acyl-2-lysoglycerophospholipids (15,34). We cannot rule out utilization of both lysolipid isomers by Lplt/Aas on the bacterial membrane.…”

Section: Discussionmentioning

confidence: 95%

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Lin¹,

Bogdanov²,

Shu³

et al. 2016

Journal of Biological Chemistry

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Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A 2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/ acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded ␣-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.

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Section: Discussionmentioning

confidence: 95%

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Lin¹,

Bogdanov²,

Shu³

et al. 2016

Journal of Biological Chemistry

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“…Phospholipid purity was monitored by TLC in CHClJCH30H/ H20 (65:24:4). Triton X-100 (Amersham), Zwittergent [3][4][5][6][7][8][9][10][11][12][13][14] (Calbiochem), and Me3CetNBr and sodium deoxycholate (Sigma) were used without further purification.…”

Section: Methodsmentioning

confidence: 99%

“…The interaction of water-soluble phospholipases with micellar structures offers a useful model system for extrinsic protein-membrane interactions. Many phospholipases have been purified to homogeneity and are available in relatively large quantities (1)(2)(3). Micelles form optically clear solutions and can be studied by a variety of conventional physical techniques (4)(5)(6)(7).…”

mentioning

confidence: 99%

Kinetic model for surface-active enzymes based on the Langmuir adsorption isotherm: phospholipase C (Bacillus cereus) activity toward dimyristoyl phosphatidylcholine/detergent micelles.

Burns¹,

El-Sayed²,

Roberts³

1982

Proc. Natl. Acad. Sci. U.S.A.

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A simple kinetic model for the enzymatic activity of surface-active proteins against mixed micelles has been developed. This model uses the Langmuir adsorption isotherm, the classic equation for the binding of gas molecules to metal surfaces, to characterize enzyme adsorption to micelles. The number of available enzyme binding sites is equated with the number of substrate and inhibitor molecules attached to micelies; enzyme molecules are attracted to the micelle due to the affinity ofthe enzyme active site for the molecules in the micelle. Phospholipase C (Bacillus cereus) kinetics in a wide variety of dimyristoyl phosphatidylcholine/detergent micelles are readily explained by this model and the assumption of competitive binding of the detergent at the enzyme active site. Binding of phospholipase C to pure detergent micelles is demonstrated by gel filtration chromatography. Phospholipase action toward phospholipid molecules in a surface is much greater than that toward monomeric substrates ["interfacial activation" (1)]. Enzyme-specific activity also depends on the matrix used to form the surface-i.e., detergent mixed micelles (8), short-chain lecithin micelles (9, 10), bilayers (11), monolayers (12). A variety of kinetic models have been applied to these phenomena. The simplest model, applied to snake venom phospholipase A2 action toward short-chain lecithins, proposes normal Michaelis-Menten kinetics and different Vm and Km values for monomeric and micellar lipid with the monomer as a competitive inhibitor of micellar lecithin (13). Another model, proposed for pancreatic phospholipase A2, accounts for interfacial activation by proposing a second site on the enzyme that "anchors" or "recognizes" surfaces (14). Different surface-active molecules can interact differentially with the two sites and hence modulate the activity. These models have not been extended in a systematic fashion to binary or more complex surfaces except in cases in which the added surface molecule is a substrate analogue. The only detailed binary component kinetic model is that ofDennis and co-workers (15). This "surface as cofactor" model was developed for phospholipase A2 and phospholipase C kinetics using Triton X-100/lecithin micelles as substrates. The model is quite complex, requiring estimation of the surface area/head-group ratio and several assumptions (16) to fit observed activities. It is based on surface association of the enzyme followed by substrate binding in the active site to form the Michaelis complex; i.e., two distinct binding steps are involved.To generalize a kinetic model for surface-active enzymes such as the phospholipases, we have examined the action of phospholipase C (Bacillus cereus) toward dimyristoyl phosphatidylcholine (Myr2PtdCho) in mixed micelles with four different detergents: Triton X-100 (nonionic), Zwittergent 3-14 (zwitterionic), deoxycholate (anionic), and trimethylcetylammonium bromide (Me3CetNBr; cationic). The data are interpreted by using a simple model based on the Langmuir adsorption is...

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“…Several snake venom enzymes have been reported to be active only in a dimeric form [5,6,23]. A protein aggregation induced by substrate micelles has been reported by Dennis et al [24] for the otherwise monomeric Nuju nuju naja phospholipase A2. Considering the stoichiometry of the lipid-protein complex of N .…”

Section: Discussionmentioning

confidence: 99%

Interaction of Phospolipase A₂ form Naja melanoleuca Sanke Venom with Meoomeric Substrate Analogs

Eijk

Verheij

Dijkman

et al. 1983

European Journal of Biochemistry

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Unlike porcine pancreatic phospholipase A,, the enzyme from Naja melanoleuca does not display biphasic kinetic behaviour at substrate concentrations around the critical inicelle concentration. This snake venom enzyme was further investigated by direct binding studies using n-tridecylphosphocholine. Binding of this substrate analog to the enzyme was monitored by using equilibrium gel filtration, equilibrium dialysis and ultraviolet difference spectroscopy. It is concluded that, in the presence of submicellar concentrations of n-tridecylphosphocholine, a lipid-protein complex is formed consisting of about 4 protein and 36 lipid molecules. CaZ + ions are required for the formation of this complex. A model is proposed which describes the formation of this type of complex. These lipidprotein aggregates are held responsible for the non-hyperbolic kinetic behaviour of the snake venom enzyme towards monomeric substrates.Phospholipase A, is widespread in Nature. The enzyme is abundant in pancreatic tissues as well as in the venoms of arthropods and snakes. Irrespective of the source, the enzymes show a high degree of homology. An identical mechanism for the hydrolysis of monomeric phospholipids has been postulated [I]. The activity of all phospholipases strongly depends on the physico-chemical state of the substrate. The enzyme displays Michaelis-Menten kinetics when acting on monomeric substrates. In the presence of certain organized lipidwater interfaces, phospholipase A, shows activities which can be three orders of magnitude higher than found for monomeric substrates. This results in a break in the velocity vs substrate concentration plots at the critical micelle concentration of the substrate [2].For the pancreatic enzymes it has been proposed by Verger et al. 131 that a particular surface region of the enzyme, called the interface recognition site (IRS), is responsible for the interaction with aggregated lipid-water interfaces.

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Cobra venom phospholipase A2: A review of its action toward lipid/water interfaces

Cited by 49 publications

References 33 publications

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Kinetic model for surface-active enzymes based on the Langmuir adsorption isotherm: phospholipase C (Bacillus cereus) activity toward dimyristoyl phosphatidylcholine/detergent micelles.

Interaction of Phospolipase A₂ form Naja melanoleuca Sanke Venom with Meoomeric Substrate Analogs

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Cobra venom phospholipase A2: A review of its action toward lipid/water interfaces

Cited by 49 publications

References 33 publications

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Kinetic model for surface-active enzymes based on the Langmuir adsorption isotherm: phospholipase C (Bacillus cereus) activity toward dimyristoyl phosphatidylcholine/detergent micelles.

Interaction of Phospolipase A2 form Naja melanoleuca Sanke Venom with Meoomeric Substrate Analogs

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Interaction of Phospolipase A₂ form Naja melanoleuca Sanke Venom with Meoomeric Substrate Analogs