Interaction of phospholipase A2 and phospholipid bilayers (original) (raw)
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Studies of crab digestive phospholipase acting on phospholipid monolayers: Activation by temperature
International Journal of Biological Macromolecules, 2019
The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. Using the classical emulsified system and the monomolecular film technique, we compared the interfacial properties of crab digestive phospholipase (CDPL) with those of the porcine pancreatic one (PPPL). A kinetic study on the surface pressure dependency of the two phospholipases was performed using monomolecular films of three substrates: di C 12-PC (1.2-dilauroyl-sn-glycerol-3phosphocholine); di C 12-PG (1.2-dilauroyl-sn-glycerol phosphoglycerol) and di C 12-PE (1.2dilauroyl-sn-glycerol phosphoethanolamine). The use of a substrate in monofilm state allows the monitoring, during the biocatalysed reactions, of several physico-chemical parameters and permits the modification of the "quality of interface". The effect of the temperature on the hydrolysis rate of these substrates was also checked. Our results show that specific activities of both phospholipases were affected by the variation of the subphase temperature. CDPL was irreversibly inactivated by p-bromo-phenacyl bromide, the specific inhibitor of secretory PLA 2 s. The hyperbolic behaviour observed was coherent with hopping mode of action, one of the two characteristic interfacial mechanisms of PLA 2 s.
Biochemistry, 1985
A number of isomeric diacylglycerophosphoholines and diacylglycero sulfates containing 0-acyl and/or S-acyl ester bonds were investigated as substrates for porcine pancreatic phospholipase A2 and its +These investigations were partly carried out under the auspices of The Netherlands Foundation for Chemical Research (SON), with financial aid from The Netherlands Organization for the Advancement of Pure Research (ZWO). Abbreviations: PLA, pancreatic phospholipase A,; proPLA, pancreatic prophospholipase A,; C, detergents, n-acyl-or (n-acylthio)glycolphosphocholines or n-acyl-or (n-acy1thio)glycol sulfates; C3 detergents, diacyl-or bis(acy1thio)glycerophosphocholines or diacyl-or bis(acy1thio)glycero sulfates. All other abbreviations used in this study have been compiled in the preceding paper (van Oort et al., 1985).
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1978
We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.
Biophysical Journal, 2010
We monitored the action of phospholipase A 2 (PLA 2) on Land D-dipalmitoyl phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA 2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the build-up of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA 2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking (SPT). Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3 µm 2 /s on the L-DPPC and 4.6 µm 2 /s on the D-DPPC monolayers. In regions enriched with hydrolysis products the diffusion dropped to ≈ 0.2 µm 2 /s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈ 30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e. enzymes were simply reflected from the gel domains back into solution.
Biochemistry, 1999
Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A 2 (PLA2) are analyzed so incremental contributions of the substitution of specific residues can be evaluated. The magnitude of the change is evaluated so a functional role in the context of the Nand C-domains of PLA2 can be assigned, and their relationship to the catalytic residues and to the i-face that makes contact with the interface. The effect of substitutions and interfacial charge is characterized by the equilibrium dissociation constant for dissociation of the bound enzyme from the interface (K d), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (K L *), and the interfacial Michaelis constants, K M * and k* cat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in k* cat is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not part of the catalytic site or the substrate binding pocket, also modulate k* cat. Interfacial anionic charge lowers K d , and induces k* cat activation through K56, K53, K119, or K120. Significant changes in K L * are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in K M * [)(k 2 +k-1)/ k 1 ] are attributed to k* cat ()k 2) and K L * ()k-1 /k 1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on K S *, and the effect of the interfacial anionic charge on k* cat. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivity, and their allosteric regulation. We suggest that the interfacial recognition region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chemical step.
Journal of Colloid and Interface Science, 2004
We present an investigation of the activity of porcine pancreatic phospholipase A 2 towards phospholipids. The phospholipids are presented in three different ways, namely as tethered vesicles, intact surface-bound vesicles, and supported planar bilayers (SPBs). The process is followed using a quartz crystal microbalance which measures both the frequency shift and the energy dissipation factor. This technique is very sensitive not only to the mass of the material deposited on the crystal, but also to its viscoelasticity. The breakdown of the phospholipid vesicles and bilayers consequently gives rise to very large signal changes. Enzyme binding is separated from vesicle hydrolysis using nonhydrolyzable ether lipids. Intact and tethered vesicles give rise to the same profile, indicating that direct immobilization of the vesicles does not affect hydrolysis significantly. The data fit well to a Voight-based model describing the change in film structure with time. Initial enzyme binding to intact vesicles is accompanied by a significant increase in layer thickness as well as a decrease in viscosity and shear modulus. This effect, which is less pronounced in SPBs, is probably mainly due to the accumulation of hydrolysis products in the vesicle prior to rupture of the vesicles and release of bound water, since it disappears when lysolipid is included in the vesicles prior to hydrolysis.
Biochemistry, 1987
In the resulting complexes the enzyme displays very high catalytic activity. In this study the interaction process was further investigated by using pancreatic phospholipases A2 of different origins and several semisynthetic mutants in which one particular amino acid residue was substituted. By use of directing binding studies with a nondegradable anionic substrate analogue and monomolecular surface film kinetics on 1,2-didecanoyl-sn-glycerol 3-sulfate, it is shown that the aggregation process is controlled by the ionization state of the side chains of the amino acid residues a t positions 6 and 17.
Biochemistry, 1983
The negatively charged detergents S-n-alkanoylthioglycol sulfates (C8, C9, and Clo) are substrates for porcine pancreatic phospholipase A2 and its zymogen. At pH 6.0 and detergent concentrations up to 0.08 X critical micelle concentration (cmc), the activities of active enzyme and zymogen are similar and very low. From 0.08 X crnc to 0.12 X cmc a tremendous increase in activity is observed for phospholipase A2, but not for the zymogen. Concomitant with this increase in activity there is a sharp rise in molecular weight I n the preceding paper (Hille et al., 1983) the interaction of porcine pancreatic phospholipase A2 and its zymogen with