Regulation of phospholipase A2 activity by the lipid-water interface: a monolayer approach

Interfacial regulation of phospholipase A2 activity on lecithin monolayers was investigated by using radioactively labeled enzyme. Labeling of the protein with 125I did not produce a change of the enzyme and protein properties as compared to the 3H fully amidinated phospholipase A2. The induction time observed during pre-steady-state kinetics reflects the rate-limiting step of the penetration of the enzyme in the interface. This penetration is reversible. However, in the surface pressure range where the enzyme is able to hydrolyze the lecithin films, the desorption of the protein from the film is slow as compared to the adsorption. Below a surface pressure of 10 dyn/cm nonspecific adsorption occurs. Using lecithins with fatty acids of different chain lengths, we have shown that the kinetics of the penetration process is governed by the packing density of the substrate molecules independent of the surface pressure. However, the steady-state surface concentration of the enzyme increases with the fatty acyl chain length of the lecithin, indicating that hydrophobic interaction occurs between phospholipase A2 and the lipid molecules at the interface. From the lecithins used pancreatic phospholipase A2 preferentially splits substrate molecules with nine carbon atoms in the acyl chain.     

Evidence that the zymogen of phospholipase A2 binds to a negatively charged lipid-water interface

Evidence is presented that the zymogen of porcine pancreatic phospholipase A2 (prophospholipase A2) interacts with a lipid-water interface provided that the interface has a net negative surface charge. Fluorescence spectroscopy and non-equilibrium gel filtration indicate that binding of prophospholipase A2 (proPLA) to mixed detergent micelles is dependent on the presence of an anionic detergent. Prophospholipase binding is accompanied by a change in the environment of the single tryptophan residue qualitatively similar to that observed when the active enzyme, phospholipase A2 (PLA), binds to micelles. In addition, the rate of tryptic activation of prophospholipase is significantly reduced in the presence of negatively-charged mixed micelles, whereas no change in rate occurs when neutral micelles are present. These observations suggest that the lack of catalytic activity of the zymogen toward organized substrates carrying a negative surface charge cannot be explained by a failure to bind at the lipid-water interface.     

Anionic center of porcine pancreatic phospholipase A2

The interaction between porcine pancreatic phospholipase A2 and low-molecular fragments of its substrate -- lecithine was studied using gel-diffusion of the enzyme in lecithin-agarose plates. When the inhibitor was added, a decrease in the magnitude of cleared areas (l/l0) around the depots filled with enzyme solution was observed. A marked decrease in l/l0 in the presence of alpha- and beta-glycerophosphates supported the statement that the cathionic center is a part of the enzyme active site SII. The potent inhibition of phospholipase activity in the presence of phosphocholine, choline, acetylcholine, thiocholine and acylthiocholines suggests the existence of an anionic center SIII in the active site. This suggestion is supported by intensive inhibition of phospholipase activity by certain, aliphatic amines. It was shown that the center is spaced in the direction of the cathionic center. SII. The main contribution to the binding of the cathionic lecithin part ("head") with the anionic center SIII is probably provided by the ion-ionic interactions.     

Solution structure of porcine pancreatic phospholipase A2.

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.     

Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.     

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.     

Hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by porcine pancreatic phospholipase A2

The hydrolysis of small unilamellar vesicles made of dipalmitoylphosphatidylcoline by pancreatic phospholipase A2 has been studied under various conditions of temperature and enzyme and substrate concentration using the following three different experimental protocols. When the enzyme was added to the substrate vesicles after being separately adjusted to the temperature of the experiments hydrolysis occurred instantaneously only in the temperature range where the lipid is known to exist in its gel phase, while above the transition range no hydrolysis occurred. Within the transition range, the time course of hydrolysis was characterized by initial very slow rate of hydrolysis (latency phase) followed by an abrupt increase in the rate after a time tau, which is a complex function of temperature and enzyme to substrate ratio. When an enzyme-substrate mixture was first preincubated below Tm and then temperature jumped to a temperature above or within the transition range, the latency phase was markedly shortened. When the temperature jump was to the transition range, this effect is observed even if Ca2+ is absent in the preincubation mixture. However, instantaneous hydrolysis was observed upon temperature jumping the mixture to a temperature high above Tm only if Ca2+ was present in the preincubation medium. In temperature-scanning experiments, hydrolysis was followed while changing the temperature of the enzyme-substrate mixture continuously. Heating an enzyme-substrate mixture from room temperature resulted in an abrupt onset of hydrolysis when the transition range was approached. These results lead us to conclude that two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicles, which for chemically pure bilayers occurs best in the gel phase. This step is followed by a Ca2+-dependent activation of the initially formed enzyme-substrate complex. The latter step only occurs under conditions where the bilayer possesses packing irregularities and probably involves a reorganization of the enzyme-substrate complex. At least one of these two steps appears to involve enzyme-enzyme interaction.     

Hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles by porcine pancreatic phospholipase A2

The interaction between dipalmitoylphosphatidylcholine large unilamellar vesicles and porcine pancreatic phospholipase A2 has been studied under a variety of conditions. It was found that the presence of large unilamellar vesicles inhibits the hydrolysis of small unilamellar vesicles at room temperature, and reaction calorimetric experiments showed that protein-lipid interactions in the absence of Ca2+ occur in the gel state with a stoichiometry of about 40 phospho-lipid molecules/protein-binding site. However, hydrolysis can be induced in the gel state under conditions of osmotic shock. On the other hand, hydrolysis is usually observed within the lipid transition temperature range, but then it occurs only after a latency phase during which the hydrolysis is very slow. The duration of this latency phase reaches a minimum near the phase transition temperature. However, if the enzyme-substrate mixture is heated from low temperatures (continuously or by a temperature jump) to a temperature within the phase transition region, hydrolysis occurs instantaneously. These results are in accordance with the conclusions of the preceding paper (Menashe, M., Romero, G., Biltonen, R. L., and Lichtenberg, D. (1986) J. Biol. Chem. 261, 5328-5333) that effective binding of the enzyme to lipid vesicles occurs relatively rapidly in the gel state and that activation of the enzyme-substrate complex requires the existence of structural irregularities in the lipid bilayer. Although hydrolysis products may have a pronounced effect on the time course of the reaction in the transition range, instantaneous hydrolysis can be induced in the phase transition region in the absence of reaction products by appropriate manipulation of the experimental conditions during which no reaction products are produced. Thus reaction products are not essential for activation of porcine pancreatic phospholipase A2. Furthermore, it is shown that the fraction of lipid hydrolyzed during the latency period is a function of the initial substrate concentration in a manner inconsistent with the proposition that the accumulation of a constant critical fraction of reaction products is the basis for activation. Comparison of the results of this study with those of the preceding paper strongly support the previously proposed reaction scheme.     

Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A2

The thioesterase activity of porcine pancreatic phospholipase A₂ has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [³H]palmitic acid from [³H]palmitoyl-acyl carrier protein.     

Thermodynamics of the binding of Ca2+ to porcine pancreatic phospholipase A2

The binding of Ca2+ to porcine pancreatic phospholipase A2 was studied by batch microcalorimetry. Enthalpies of binding at 25 degrees C were determined as a function of Ca2+ concentration in buffered solutions at pH 8.0 using both the Tris-HCl and Hepes-NaOH buffer systems. The calorimetric results indicate that protons are released on calcium binding and that in addition to the binding of the active-site calcium, there appears to be weak binding of a second Ca2+. Results from potentiometric titrations indicate that this proton release on binding Ca2+ arises from a change in pK of a histidine(s) functional group. The thermodynamic functions delta G0, delta H0 and delta S0 for calcium binding to phospholipase A2 have been determined. These results are compared with literature data for Ca2+ complex formation with some small molecules and also the protein troponin-C.     

Studies on the role of methionine in porcine pancreatic phospholipase A2.

The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.     

Multiple forms of porcine pancreatic phospholipase A2: Isolation and specificity

Porcine pancreatic phospholipase A₂ was purified from commercial pancreatin by a method involving heat denaturation, trichloroacetic acid precipitation, and DEAE-cellulose chromatography. Assaying the eluate of the chromatography step by a new titrimetric method using vegetable lecithin-albumin emulsion as the substrate, several species of phospholipase A were found. Some of these went undetected when the conventional egg yolk emulsion assay was used. Two phospholipases A₂ were isolated in a homogeneous form and shown to have similar chemical and physical properties. Catalytic specificity of the two enzymes differs remarkably toward lecithins in different emulsified states.     

NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A2.

Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine, with competitive inhibitors derived from the following general structure: [formula: see text] X and Y are alkyl chains with various 'reporter groups'. The interactions between the inhibitor and the enzyme were localized by comparison of 2-D nuclear Overhauser effect spectra using protonated and selectively deuterated inhibitors, and inhibitors with groups having easily identifiable chemical shifts. These experiments led us to the following conclusions for the phospholipase A2/inhibitor/micelle complex: i) the His48 C2 ring proton is in close proximity to both the amide proton and the methylene protons at the sn-1 position of the glycerol skeleton of the inhibitor, ii) the acyl chain of the inhibitor at the sn-2 position makes hydrophobic contacts near Phe5, Ile9, Phe22 and Phe106; iii) no interactions between the acyl chain at the sn-1 position and the protein could be identified. Comparison of our results on the enzyme/inhibitor/micelle ternary complex with the crystal structure of the enzyme-inhibitor complex shows that the mode of inhibitor binding is similar. However, in several cases we found indications that the hydrophobic chains of the inhibitors can have multiple conformations.     

Activation of phospholipase A2 by ternary model membranes

Formation of liquid-ordered domains in model membranes can be linked to raft formation in cellular membranes. The lipid stoichiometry has a governing influence on domain formation and consequently, biochemical hydrolysis of specific lipids has the potential to remodel domain features. Activation of phospholipase A₂ (PLA₂) by ternary model membranes with three components (DOPC/DPPC/Cholesterol) can potentially change the domain structure by preferential hydrolysis of the phospholipids. Using fluorescence microscopy, this work investigates the changes in domain features that occur upon PLA₂ activation by such ternary membranes. Double-supported membranes are used, which have minimal interactions with the solid support. For membranes prepared in the coexistence region, PLA₂ induces a decrease of the liquid-disordered (L_d) phase and an increase of the liquid-ordered (L_o) phase. A striking observation is that activation by a uniform membrane in the L_d phase leads to nucleation and growth of L_o-like domains. This phenomenon relies on the initial presence of cholesterol and no PLA₂ activation is observed by membranes purely in the L_o phase. The observations can be rationalized by mapping partially hydrolyzed islands onto trajectories in the phase diagram. It is proposed that DPPC is protected from hydrolysis through interactions with cholesterol, and possibly the formation of condensed complexes. This leads to specific trajectories which can account for the observed trends. The results demonstrate that PLA₂ activation by ternary membrane islands may change the global lipid composition and remodel domain features while preserving the overall membrane integrity.