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 kinetic parameters for the steady-state rate of hydrolysis of egg phosphatidylcholine in multilamellar vesicles by bee venom phospholipase A2 are measured in the presence of 27 alkanols and several organic solvents. In general, small nonpolar solutes like enflurane, tetrahydrofuran, benzene, chloroform and diethylether do not promote the hydrolysis of multilamellar vesicles. The rate of hydrolysis shows a biphasic dependence upon the alkanol concentration for all higher (C5-C9) alcohols examined, i.e., an optimal rate of hydrolysis is observed at a characteristic concentration for each alcohol. The alkanol to lipid mole ratio (D/L ratio) in the bilayer at the peak activating concentration of an alkanol was computed from its bilayer/water partition coefficient. The branched chain alcohols induce peak activation of hydrolysis at lower D/L ratios in the bilayer than the corresponding straight chain analogs. Similarly, the longer chain n-alkanols at peak activating concentration have a lower D/L ratio than the corresponding lower alcohols. Both the Km and Vm for phosphatidylcholine increase as a function of the chain length of the activating alcohol. These kinetic parameters also depend upon the position of the substituents on the activating alcohols. Both the D/L ratio and Vm for an alcohol are found to change with the cross-sectional area of the activating alcohol across its long axis: alcohols with a more asymmetric cross-section exhibit higher Vm and a lower D/L ratio. Such correlations of Vm and D/L ratio with the molecular parameters of the alkanols are interpreted to suggest that the accessibility of the substrate molecule in the bilayer to the phospholipase is modulated by the free space introduced by the alkanols in the bilayer. Effect of tetradecane derivatives and A2C (a membrane fluidizing agent) on the phase transition characteristics of DPPC bilayers, and their susceptibility to phospholipase A2 from bee venom and pig pancreas is also reported. These solutes cause a broadening of the transition profile and reduce the size of the cooperative unit and the enthalpy of transition. These effects depend upon the mole fraction of a solute in the bilayer; however, equal concentrations of these solutes do not induce equal response. Susceptibility of the modified bilayers to phospholipase A2 depends not only upon the structure of the solute but also upon the source of the enzyme. The data show that the activity of the membrane-bound enzyme is modulated to different extents by different solutes, and the bilayer perturbing ability of these solutes may be related to the asymmetry of their cross-sectional area and to the free space introduced by the alkanols in a bilayer.
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PMID:Intrinsic differences in the perturbing ability of alkanols in bilayer: action of phospholipase A2 on the alkanol-modified phospholipid bilayer. 719 Oct 9

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 degrees C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97-106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis-Menten function dependent on the catalytic properties of the enzyme (kcat approximately equal to 9200 min-1, Km approximately equal to 0.12 mM), then level off to a slower rate determined by the desorption equilibrium constant (KD approximately equal to 0.01 mM) and desorption rate constant (kD approximately equal to 0.15 min-1). The relaxation time, tau, for the transition to the desorption-limited reaction is approximately 0.5 min; this large value of tau probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lysophosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decrease to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.
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PMID:Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine. 745 86

The rate of hydrolysis of phosphatidylcholine bilayers by soluble phospholipase A2 (PLA2) is greatly enhanced by the presence in the bilayer of a threshold mole fraction of the reaction products: fatty acid and lysophosphatidylcholine (lyso-PC). The threshold requirement of these products appears to vary as a function of vesicle and calcium concentration. To further identify the roles of myristic acid, lyso-PC, and calcium in promoting optimal PLA2 activity, we have quantified the various interactions among these components and dimyristoylphosphatidylcholine large unilamellar vesicles. The bilayer/water partition coefficient for myristic acid was obtained by competition of vesicles for the binding of the fatty acid to an acrylodan conjugate of an intestinal fatty acid binding protein as monitored by the acrylodan fluorescence emission spectrum. The partition coefficient for lyso-PC was obtained by a similar procedure using the tryptophan emission spectrum of bovine serum albumin. The effect of calcium concentration on these interactions was also quantified. These results were incorporated into an empirical model to describe the threshold requirements for these products in the bilayer. This information is vital for elucidating the mechanism of activation of PLA2 by the hydrolysis products.
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PMID:Quantification of the interactions among fatty acid, lysophosphatidylcholine, calcium, dimyristoylphosphatidylcholine vesicles, and phospholipase A2. 785 76

A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. 801 13

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 effect of detergents on the overall catalytic turnover by secreted phospholipase A2 (PLA2) on codispersions of the substrate phospholipid is characterized. The overall rate of interfacial catalytic turnover depends on the effective substrate "concentration" (mole fraction) that the bound enzyme "sees" at the interface. Therefore, besides the intrinsic catalytic turnover rate determined by the Michaelis-Menten cycle in the interface [Berg et al. (1991) Biochemistry 30, 7283], two other interfacial processes significantly alter the overall effective rate of hydrolysis: first, the fraction of the total enzyme at the interface; second, the rate of replenishment of the substrate. At low mole fractions (< 0.3), bile salts promote the binding of pig pancreatic PLA2 to zwitterionic vesicles, and the rate of hydrolysis increases with the fraction of the enzyme in the interface. At higher (> 0.3) mole fractions of the detergent, the bilayer is disrupted, and the rate of hydrolysis decreases by more than a factor of 10. The detergent-dependent decrease in the rate of hydrolysis of the sn-2-oxyphospholipids is much larger than that of sn-2-thiophospholipid, and therefore the element effect (O/S ratio) decreases from about 10 in bilayers to less than 2 in mixed micelles. This loss of the element effect in mixed micelles shows that the chemical step is no longer rate-limiting during the hydrolysis of mixed micelles formed by the disruption of vesicles by the detergent. Such effects were observed with phospholipase A2 from several sources acting on substrates dispersed in a variety of detergents including bile salts, 2-deoxylysophosphatidylcholine, and Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The chemical step is not rate-limiting during the hydrolysis by phospholipase A2 of mixed micelles of phospholipid and detergent. 834 32

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

The activity of soluble phospholipase A2 to hydrolyze phosphatidylcholine vesicles increases abruptly after a lag time of several minutes. The onset of this apparent activation event probably results from the accumulation of a threshold mole fraction of the hydrolysis products (lysolecithin and fatty acid) in the bilayer. One important observation relevant to the mechanism of this activation process is the biphasic dependence of the lag time on vesicle concentration. To test whether this dependence can be attributed entirely to the strength of partitioning of the lysolecithin into the phosphatidylcholine bilayer, we estimated the apparent partition coefficient of lysophospholipid in the membrane of phosphatidylcholine vesicles. Based on competition between bovine serum albumin and the vesicles for the lysophospholipid, we estimated the partition coefficient to be about 5.10(-7) for palmitoyl lipids at 39 degrees C and about 9.10(-7) for myristoyl lipids at 22 degrees C. These values were able to rationalize the behavior of the lag time with dipalmitoylphosphatidylcholine vesicles, but they were unable to predict the behavior with dimyristoylphosphatidylcholine. Therefore, it appears that the complete dependence of the lag phase on vesicle concentration must be explained by additional means such as the possible contribution of nascent fatty acid or previously proposed kinetic activation mechanisms.
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PMID:Quantification of the interaction of lysolecithin with phosphatidylcholine vesicles using bovine serum albumin: relevance to the activation of phospholipase A2. 850 38

1. The patch-clamp technique was used to characterize chloride channels from the apical membranes of bovine tracheal epithelial cells. Application of GTP gamma S or NaF to excised patches revealed the existence of a novel type of Cl- channel regulated by G-proteins in a membrane-delimited manner. 2. The channel had a linear current-voltage relationship, with a conductance of 100-120 pS. Its open probability was independent of voltage. 3. The channel was highly anion selective (permeability ratio, PNa/PCl = 0.06 +/- 0.04) and had the halide permeability sequence: I- > Br- > or = Cl- > F-, corresponding to the Eisenman I sequence. This suggested that neither ionic size nor diffusion rate determined ion permeation through the channel. 4. The mole fraction behaviour was studied using fluoride and chloride ions. Mixtures of ions produced currents that would be expected from the linear combination of the two ions acting independently, indicating relatively simple permeation through the pore and compatible with a single ion binding site. 5. The channel was inhibited by the stilbene disulphonates SITS (4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulphonic acid) and DNDS (4,4'-dinitrostilbene-2,2'-sulphonic acid). SITS introduced voltage dependence to channel gating and indicated the possible involvement of lysine residues in the channel permeation pathway. 6. NaF was unable to activate Cl- channels in the presence of the aluminum chelator, deferoxamine mesylate. This indicates that Al3+ ions play an important role in chloride channel activation by fluoride. NaF activation was not dependent on the presence of calcium ions. 7. The channel was insensitive to alkaline phosphatase and to the specific inhibitors of protein phosphatase types I and 2A, okadaic acid and calyculin A. 8. The channels could be activated by GTP gamma S or by NaF in the presence of the phospholipase A2 inhibitor quinacrine, indicating that this enzyme is not involved in channel regulation.
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PMID:Characterization and regulation of a chloride channel from bovine tracheal epithelium. 858 18

Hemorrhagic factor I (LHF-I) was previously purified from the venom of the bushmaster snake (Lachesis muta muta). In terms of biochemical and immunological properties, LHF-I is a glycoprotein (mol. wt 100,000, pI 4.7) consisting of two subunits; it loses its activity following mercaptoethanol treatment. LHF-I contains 0.7 g-atom zinc and 1.2 g-atom calcium per mole protein. The hemorrhagic and the proteinase activities are inhibited by EDTA; subsequent addition of Ca2+ or Mg2+ does not reverse the EDTA-induced inhibition of the hemorrhagic activity. The metalloenzyme does not hyrolyze arginine esters and is devoid of phospholipase A2 activity. It hydrolyzes the A alpha- > B beta-chain of fibrinogen without clot formation and hydrolyzes selectively the alpha-chain of fibrin, leaving the B beta- and tau-chains unaffected. Antibodies to the hemorrhagic factor in bushmaster venom were produced by immunizing rabbits with the purified protein. The antibody was purified by protein-A affinity chromatography. This antibody was also used to screen other Crotalinae venom samples for immunologically similar epitopes by ELISA assay. The purified antibody reacted only with LHF-I and two samples of bushmaster venom from different geographical locations.
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PMID:Characterization of a hemorrhagic factor, LHF-I, isolated from the bushmaster snake (Lachesis muta muta) venom. 886 22


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