Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The initial rate of hydrolysis of large unilamellar vesicles of dipalmitoylphosphatidylcholine by phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus is small and elevates gradually until it suddenly increases by a factor of 10 to 1000 depending on the experimental conditions. This abrupt onset of high enzyme activity appears to be correlated to a specific mole fraction of reaction product at which point a cooperative compositional phase transition in the bilayer occurs. Five models that describe the activation process in terms of its being coupled to the putative product-induced lipid transition are presented. These models include one in which the lipid structure enhances the affinity of enzyme binding to the bilayer surface, two in which the equilibrium position between an active and an inactive form of the enzyme-substrate complex is altered, and two in which the rate of a quasi-irreversible spontaneous activation process is increased. Whether the active form of the enzyme is a monomer or dimer is also considered in the last two pairs of models. Computer simulations of time courses for the different models show how a set of four experimental observables distinguishes qualitatively among them. Comparison of the experimental behavior with the computer-simulated behavior of the observables for each model indicates that activation of phospholipase A2 on the lipid surface involves formation of an enzyme dimer which spontaneously converts to an active form. The active enzyme persists in the active state as it exchanges between vesicles. This model of activation is similar to that proposed previously for activation of porcine pancreatic phospholipase A2.
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PMID:Molecular details of the activation of soluble phospholipase A2 on lipid bilayers. Comparison of computer simulations with experimental results. 159 46

Site-directed mutagenesis was used to probe the structural and functional roles of two highly conserved residues, Tyr-52 and Tyr-73, in interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overproduced in Escherichia coli). According to crystal structures, the side chains of these two active site residues form H-bonds with the carboxylate of the catalytic residue Asp-99. Replacement of either or both Tyr residues by Phe resulted in only very small changes in catalytic rates, which suggests that the hydrogen bonds are not essential for catalysis by PLA2. Substitution of either Tyr residue by nonaromatic amino acids resulted in substantial decreases in the apparent kcat toward 1,2-dioctanoyl-sn-glycero-3-phosphocholine (DC8PC) micelles and the v(o) (turnover number at maximal substrate concentration, i.e., mole fraction = 1) toward 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DC14PM) vesicles in scooting mode kinetics [Berg, O. G., Yu, B.-Z., Rogers, J., & Jain, M. K. (1991) Biochemistry 30, 7283-7297]. The Y52V mutant was further analyzed in detail by scooting mode kinetics: the E to E* equilibrium was examined by fluorescence; the dissociation constants of E*S, E*P, and E*I (KS*, KP*, and KI*, respectively) in the presence of Ca2+ were measured by protection of histidine-48 modification and by difference UV spectroscopy; the Michaelis constant KM* was calculated from initial rates of hydrolysis in the absence and presence of competitive inhibitors; and the turnover number under saturating conditions (kcat, which is a theoretical value since the enzyme may not be saturated at the interface) was calculated from the vo and KM* values. The results indicated little perturbation in the interfacial binding step (E to E*) but ca. 10-fold increases in KS*, KP*, KI*, and KM* and a less than 10-fold decrease in kcat. Such changes in the function of Y52V are not due to global conformational changes since the proton NMR properties of Y52V closely resemble those of wild-type PLA2; instead, it is likely to be caused by perturbed enzyme-substrate interactions at the active site. Tyr-73 appears to play an important structural role. The conformational stability of all Tyr-73 mutants decreased by 4-5 kcal/mol relative to that of the wild-type PLA2. The proton NMR properties of Y73A suggested significant conformational changes and substantially increased conformational flexibility. These detailed structural and functional analyses represent a major advancement in the structure-function study of an enzyme involved in interfacial catalysis.
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PMID:Phospholipase A2 engineering. Structural and functional roles of highly conserved active site residues tyrosine-52 and tyrosine-73. 163 53

The miscibility of 1,3-dioleoylglycerol (DOG) with 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) dispersed in excess buffer was characterized by physical and enzymatic methods. Thermograms for all SOPC-DOG mixtures exhibit a transition at 5.3 degrees C. Above 0.25 mole fraction of DOG, metastability is observed; after the first scan, a second peak appears at 23.4 degrees C which corresponds to the chain melting of pure DOG. This suggests that a complex or preferred packing array is formed which has a DOG mole fraction of 0.25 (XC). Bilayer morphology is maintained in the metastable state up to 0.8 mole fraction of DOG. Above 0.8, a novel, nonlamellar phase is formed. Fluorescence polarization of 1,6-diphenylhexatriene shows that, relative to SOPC alone, there is little change in the order of the acyl chains up to Xc followed by a large decrease above Xc. Similar results were obtained using POPC. Miscibility was also studied in lipid films at the argon-buffer interface. Isothermal phase diagrams for the mixtures at 15 and 24 degrees C exhibited phosphatidylcholine-DOG complex formation, a region of phosphatidylcholine and complex coexistence, and a region of complex and DOG miscibility. The mole fractions of DOG in the complex (Xc) range from 0.24 to 0.27. Porcine pancreatic phospholipase A2 and pancreatic lipase plus colipase were used as probes of the surface in both the monolayer and bilayer systems. In both systems and with both enzymes, substrate hydrolysis increased abruptly with increasing DOG.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enzymatic and physical characterization of diacylglycerol-phosphatidylcholine interactions in bilayers and monolayers. 270 55

Experimental conditions are described for simultaneous purification of three forms of lipocortin (lipocortin I, lipocortin II and lipocortin-85) from bovine lung. The procedure yields milligram quantities of all three lipocortins. Using antisera against lipocortin I and lipocortin II, purified proteins show no cross contaminations. All forms of lipocortin exhibit equal potency as in vitro bovine pancreatic phospholipase A2 inhibitors. Protein kinase C catalyzes the in vivo incorporation of about 1.0, 0.7 and 0.4 mole of phosphate per mole of lipocortin I (p35), lipocortin II (p36) and lipocortin-85 (p36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While lipocortin I is phosphorylated on threonine residues, lipocortin II and lipocortin-85 are phosphorylated on serine residues.
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PMID:Purification of three forms of lipocortin from bovine lung. 295 2

Binding of pig pancreatic phospholipase A2 to ternary codispersions of diacylphosphatidylcholine/lysophosphatidylcholine/fatty acid (100:22:22, mole ratio) is monitored by the increase in intrinsic fluorescence intensity of the single tryptophan residue. The fluorescence is quenched by the brominated fatty acid components in the ternary codispersions. The quenching efficiency is in the order: 11,12-dibromo- greater than 9,10-dibromo- greater than 6,7-dibromo- greater than 2-bromo fatty acid. The quenching efficiency of the 9,10-brominated derivatives of the three components in the ternary codispersions is in the order diacylphosphatidylcholine greater than fatty acid greater than lysophosphatidylcholine. Two isomers of diacylphosphatidylcholine with 9,10-dibromo substituents on chain 1 or 2 are equally efficient quenchers. While succinimide also quenches the fluorescence of the free and the membrane bound enzyme, the tryptophan residue in both systems is not accessible to 1-methylnicotinamide. These results are rationalized by a hypothesis that the acyl chains of the substrate interacts with the tryptophan residue of pig pancreatic phospholipase A2, which is readily accessible to water soluble neutral quenchers both in the free and the bound state.
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PMID:The environment of tryptophan in pig pancreatic phospholipase A2 bound to bilayers. 397 97

The reaction progress curve for the action of pig-pancreatic phospholipase A2 on dimyristoylphosphatidylcholine vesicles is characterized under a variety of conditions. The factors that regulate the rate of hydrolysis during the presteady-state phase determine the latency period. The results demonstrate that the accelerated hydrolysis following the latency phase of the reaction progress curve is due to the product-assisted binding of the enzyme to the substrate bilayer by chaning the number of bindings sites and therefore the binding equilibrium. A critical mole fraction of products appears to be formed in the substrate bilayers before the steady-state phase of hydrolysis begins. The latency phase shows a minimum at the phase-transition temperature of the substrate vesicles; however, we did not observe a significant binding of the enzyme to pure substrate bilayers even at the phase-transition temperature. The rate of binding of the enzyme is found to be fast and the rate of desorption of the bound enzyme is very slow compared to the latency phase. The rate of redistribution of products between substrate bilayers is rather slow. These observations demonstrate that during the latency phase of the action of phospholipase A2, a critical mole fraction of products is formed in the substrate bilayer.
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PMID:Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles. 710 29

Carboxylase groups in bovine pancreatic phospholipase A2 were modified using a water-soluble carbodiimide and semicarbazide. At pH 5.5 rapid modification of 13 out of the total of 15 carboxylates occurred, leaving Asp-39 and Asp-99 unmodified. Subsequent modification with radioactive semicarbazide at pH 3.5 resulted in the labelling of Asp-39. In the modified proteins both Ca2+ binding and enzymatic activity are completely lost. Modification of the enzyme at pH 5.5 in the presence of Ca2+ ions yielded a protein with three unmodified carboxylates which is capable of binding Ca2+ ions, while 15% residual activity was found. A second reaction, using [14C]semicarbazide at pH 5.5 in the absence of Ca2+, leads to the incorporation of one mole of [14C]semicarbazide/mole of protein with loss of enzymatic activity and Ca2+ binding. The label was found to be attached to Asp-49, demonstrating the essential role of Asp-49 in Ca2+ binding. Studies on the pH dependence of the Ca2+ binding to the protein suggested that Asp-49 has an apparent pK of 5.2.
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PMID:Modification of carboxylate groups in bovine pancreatic phospholipase A2. Identification of aspartate-49 as Ca2+-binding ligand. 720 11

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.
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PMID:Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers. 811 Aug 7

Sporidesmin, a mycotoxin from Pithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A2 (PLA2). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations < 10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (approximately 30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA2 kinetics are consistent with their ability to perturb bilayer organisation.
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PMID:Interaction of sporidesmin, a mycotoxin from Pithomyces chartarum, with lipid bilayers. 830 34

The rate and equilibrium parameters for the interfacial catalysis by recombinant human nonpancreatic secreted phospholipase A2 were determined. Results show that the enzyme binds to anionic interfaces with considerably higher affinity than to zwitterionic interfaces. The extent of hydrolysis per enzyme on anionic vesicles in the processive scooting mode shows that the enzyme is fully catalytically active as a monomer. Among several secreted phospholipases A2 tested, the human nonpancreatic secreted enzyme is unique in its ability to undergo slow intervesicle exchange either by dissociation from the interface followed by binding to a different vesicle or by promoting the fusion of vesicles. The equilibrium dissociation constants for calcium, substrate analogs, reaction products, and several competitive inhibitors bound to the enzyme at the interface were determined by monitoring the ligand-conferred protection of the active site histidine residue from alkylation by phenacyl bromide. The interfacial Michaelis-Menten parameters were determined from the analysis of the entire reaction progress curve and also by monitoring the effect of competitive inhibitors on the initial rate of hydrolysis in the scooting mode. The interfacial Michaelis constant (KM*) for the substrate 1,2-dimyristoylglycero-sn-3-phosphomethanol was determined to be considerably above the maximal attainable mole fraction of unity for the substrate in the bilayer. Substrate specificity studies show that the enzyme does not significantly discriminate between phospholipids that differ in the type of polar head group or in the degree of unsaturation of the fatty acyl chains. Competitive inhibitors are described that display a high degree of selectivity for binding to the nonpancreatic versus pancreatic phospholipase A2. The kinetic properties of the human nonpancreatic secreted phospholipase A2 suggest that the enzyme has evolved to hydrolyze substrates at anionic interfaces and at high calcium concentrations.
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PMID:Human nonpancreatic secreted phospholipase A2: interfacial parameters, substrate specificities, and competitive inhibitors. 842 68


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