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.     

Kinetic and inhibition studies of phospholipase A2 with short-chain substrates and inhibitors

The action of the phospholipases A2 (PLA2s) from Naja naja naja, Naja naja atra, and Crotalus atrox venoms as well as the enzyme from porcine pancreas on a number of short-chain, water-soluble substrates was studied. The inhibition of these enzymes by short-chain phosphonate- and thiophosphonate-containing phospholipid analogues was also examined. The kinetic patterns observed for the action of the venom PLA2s on substrates containing phosphocholine head groups all deviated from a classical Michaelis-Menten-type behavior. With a substrate containing an anionic head group, the kinetic pattern observed was more normal. In contrast, Michaelis-Menten-type behavior was observed for the action of the porcine pancreatic PLA2 acting on all of the substrates studied. A short-chain phospholipid analogue in which the enzyme-susceptible ester was replaced with a phosphonate group was found to be a tight-binding inhibitor of the venom PLA2s with IC50 values that were some 10(4)-10(5)-fold lower than the concentration of substrate used in the assay. The degree of inhibition was found to depend dramatically on the stereochemical arrangement of substituents in the inhibitor which strongly suggests that the inhibitors are binding directly to the active site of the PLA2s. By comparison, the phosphonate analogue functioned as a poor inhibitor of the porcine pancreatic PLA2. Direct inhibitor binding studies indicated that the short-chain phosphonate inhibitor bound weakly to the venom enzymes in the absence of the short-chain substrates. Several other unusual features of the inhibition were also observed. The data are interpreted in terms of a model in which the enzyme and substrate form a lipid-protein aggregate at substrate concentrations below the critical micelle concentration (cmc). Possible reasons for the selective binding of the inhibitor to the enzyme-substrate microaggregate are discussed.     

Localization of phospholipase A2 in normal and ras-transformed cells

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.     

Catalytic Ca2+-binding site of pancreatic phospholipase A2: laser-induced Eu3+ luminescence study

7F0----5D0 excitation spectroscopy of Eu3+ has been used to study the catalytic Ca2+-binding site of pancreatic phospholipases A2. Eu3+ binds competitively with Ca2+ to the enzyme with retention of about 5% of the activity found with Ca2+. The dissociation constants for the Eu3+-enzyme complexes of bovine phospholipase A2 and porcine isophospholipase A2 are 0.22 mM and 0.16 mM, respectively. Results obtained with the porcine phospholipase A2 at neutral pH indicate aggregation of this enzyme at protein concentrations above 0.18 mM. The Eu3+ bound at the catalytic site of pancreatic phospholipase A2 is coordinated to four or five water molecules, which, in conjunction with binding constant data, suggests the involvement of two or three protein ligands. Addition of a monomeric substrate analogue to the enzyme-Eu3+ complex results in the loss of an additional water molecule from the first coordination sphere of the bound Eu3+. This result suggests an interaction between the negative charge of the polar head group of the substrate analogue and the Eu3+. Binding of the enzyme-Eu3+ complex to micelles results in a nearly complete dehydration of the Eu3+ bound to the catalytic center. In the phospholipase A2-Eu3+-micelle complex, only one H2O molecule is coordinated to Eu3+. This dehydration at the active site of phospholipase A2 in the protein-lipid complex can be an important reason for the enhanced activity of this enzyme at lipid-water interfaces.     

Competitive inhibition of lipolytic enzymes. V. A monolayer study using enantiomeric acylamino analogues of phospholipids as potent competitive inhibitors of porcine pancreatic phospholipase A2.

For the first time, we have shown that a stereospecific interaction occurs between porcine pancreatic phospholipase A2 and a monomolecular film of amidophospholipid used as inhibitor. Direct binding experiments, using radiolabelled phospholipase A2, showed that 13 times more enzyme was bound to phospholipid films of the L series by comparison with films of the D series. These results were confirmed by indirect binding studies using re-spreading experiments. Kinetic studies of the porcine pancreatic PLA2, using enantiomeric acyl-amino phospholipid analogues, have shown that: (1) inhibitors of the L series are more potent than inhibitors of the D series, (2) inhibitors having a negative charge are more potent than zwitterionic inhibitors, (3) inhibitory power values are greater when evaluated in micellar system than in a the monolayer system, (4) the inhibitory power increases continuously with surface pressure.     

Porcine pancreatic phospholipase A2: sequence-specific 1H and 15N NMR assignments and secondary structure

The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior.     

Site-specific epsilon-NH2 monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating "membrane-bound" form

Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.     

Secretion of the proenzyme and active bovine pancreatic phospholipase A2 enzyme by Saccharomyces cerevisiae: design and use of a synthetic gene

We have developed a phospholipase A2(PLA2)-producing system using Saccharomyces cerevisiae. A 456-bp synthetic DNA fragment was constructed encoding bovine pancreatic phospholipase A2 (proPLA2; zymogen) along with the signal sequence of dog pancreatic PLA2. Yeast-preferred codons were chosen and unique restriction enzyme sites were incorporated. 22 oligodeoxynucleotides that varied in size from 33 to 48 nucleotides were chemically synthesized and assembled into the DNA fragment, which was then placed under the control of the yeast acid phosphatase repressible promoter. The resulting plasmid, transformed into S. cerevisiae, directed the synthesis of about 2.8 micrograms/ml of PLA2, most of which was secreted into the culture fluid. The secreted PLA2 comprised 18 to 26% of active enzyme, the remainder being proenzyme. Both had the expected N-terminal amino acid sequences, indicating that the yeast accurately released the signal peptide and the activation peptide (N-terminal heptapeptide of proPLA2). The specific activity of PLA2 thus produced is the same as that of the authentic bovine enzyme.     

Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.     

1H-NMR and protection studies of interactions between ligands and bovine pancreatic phospholipase A2

A number of long-chain amines and naphthylamine sulfonates have been studied for their ability to inhibit bovine pancreatic phospholipase A2 (PLA2) and to protect PLA2 against alkylation of the active site histidine by p-bromophenacyl bromide. Their areas of interaction on the enzyme were further delineated using observations of chemical shift changes of assigned aromatic signals in the 1H-NMR spectrum of PLA2, while the bound conformations of two amine inhibitors were revealed using transferred nuclear Overhauser effects. The alkyl amines bind rather non-specifically on the surface of the enzyme, over the active site cleft and the interface recognition site.     

Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes

We described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the "type II" viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.     

Differential interfacial and substrate binding modes of mammalian pancreatic phospholipases A2: a comparison among human, bovine, and porcine enzymes.

To identify the residues essential for interfacial binding and substrate binding of human pancreatic phospholipase A2 (hpPLA2), several ionic residues in the putative interfacial binding surface (R6E, K7E, K10E, and K116E) and substrate binding site (D53K and K56E) were mutated. Interfacial affinity of these mutants was measured using anionic polymerized liposomes, and their enzymatic activity was measured using various substrates including phospholipid monomers, zwitterionic and anionic micelles, and anionic polymerized mixed liposomes. Similar mutations (R6E, K10E, K56E, and K116E) were made to porcine pancreatic phospholipase A2 (ppPLA2), and the properties of mutants were measured by the same methods. Results indicate that hpPLA2 and ppPLA2 have similar interfacial binding mechanisms in which cationic residues in the amino terminus and Lys-116 in the carboxy terminus are involved in binding to anionic lipid surfaces. Small but definite differences between the two enzymes were observed in overall interfacial affinity and activity and the effects of the mutations on interfacial enzyme activity. The interfacial binding of hpPLA2 and ppPLA2 is distinct from that of bovine pancreatic phospholipase A2 in that Lys-56 is involved in the interfacial binding of the latter enzyme. The unique phospholipid headgroup specificity of hpPLA2 derives from the presence of Asp-53 in the substrate binding site. This residue appears to participate in stabilizing electrostatic interactions with the cationic ethanolamine headgroup, hence the phosphatidylethanolamine preference of hpPLA2. Taken together, these studies reveal the similarities and the differences in the mechanisms by which mammalian pancreatic phospholipases A2 interact with lipid aggregates and perform interfacial catalysis.     

Presence of a phospholipase A2 inhibitor in porcine serum

Porcine pancreatic phospholipase A2 (PLA2) was immobilized to Sepharose 4B and porcine serum was passed through this affinity column. Bound substances were eluted by an EDTA-containing buffer and fractionated in a Sepharose 6B column. A single protein peak of the eluate from the latter column was found to inhibit PLA2 activity in a dose-dependent manner in an assay system using radioactive lecithin as a substrate and porcine pancreatic PLA2 as the enzyme source. The serum fraction containing the PLA2 inhibitory protein(s) (PIP) appeared inhomogeneous on SDS-polyacrylamide gel electrophoresis with two major bands close to each other, corresponding to a molecular weight of approximately 60,000. It was concluded that PIP might act as a protective principle against autodigestion in acute pancreatitis and other inflammatory diseases as well as playing a regulatory role in prostaglandin metabolism.     

Inhibitory effect of platinum and ruthenium bipyridyl complexes on porcine pancreatic phospholipase A2

Pancreatic phospholipase A(2) (PLA(2)) plays an important role in cellular homeostasis as well as in the process of carcinogenesis. Effects of metallo-drugs used as chemotherapeutics on the activity of this enzyme are unknown. In this work, the interaction between porcine pancreatic PLA(2) and two selected transition metal complexes--tetrachloro(bipyridine) platinum(IV) ([PtCl(4)(bipy)]) and dichloro (bipyridine) ruthenium(III)chloride ([RuCl(2)(bipy)(2)]Cl)--was studied. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence spectroscopy have been used to analyse the enzyme activity in the absence and presence of metal complexes and to verify potential binding of these drugs to the enzyme. The tested metal complexes decreased the activity of phospholipase A(2) in an uncompetitive inhibition mode. A binding of the ruthenium complex near the active site of the enzyme could be evidenced and possible modes of interaction are discussed.     

Hydrolysis of 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero- 3-phospholipids by phospholipase A2: effect of the polar head-group

The effect of the phospholipid polar head-group on the porcine pancreatic phospholipase A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) reaction was studied using 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3- phosphatidylcholine, -ethanolamine, -glycerol, -monomethylester and -serine as substrates. Except for the monomethylester analogue, which was maximally activated by 3.5 mM CaCl2, maximal enhancement of hydrolysis of the other pyrenephospholipids was obtained at 2 mM Ca2+. Sodium cholate inhibited hydrolysis of the ethanolamine and serine lipids, whereas a slight (1.4-2.0-fold) activation was observed for the -choline, -glycerol and -monomethylester derivatives. Arrhenius plots of hydrolysis of pyrenephospholipids by porcine pancreatic phospholipase A2 revealed no discontinuities, thus indicating the absence of phase transition for these lipids in the temperature range 15-45 degrees C. Specific activities of porcine and bovine pancreatic, porcine intestinal and snake venom (Crotalus atrox) phospholipases A2 towards pyrenephospholipid liposomes were then compared. Whereas the snake venom phospholipase A2 preferred phosphatidylcholine as a substrate, the other phospholipases A2 preferred acidic phospholipids in the order monomethylester greater than or equal to glycerol greater than or equal to serine.     

Adaptability of restrained molecular dynamics for tertiary structure prediction: application to Crotalus atrox venom phospholipase A2

In order to assess the adaptability and/or applicability of the restrained molecular dynamics (RMD) simulation for building a possible tertiary structure of a protein from the X-ray crystal structure of a family reference protein, the tertiary structure prediction of Crotalus atrox venom phospholipase A2 (PLA2) was attempted based on the X-ray crystal structure of bovine pancreatic PLA2. For the formation of secondary and tertiary structures from the fully extended starting structure, the RMD simulation with interatomic distance restraints and torsion angle restraints, which were derived from homologous amino acid sequence regions in the reference protein, was carried out until the molecular system was fully equilibrated. The predicted tertiary structure of C. atrox venom PLA2 was compared with its X-ray crystal structure, and furthermore the utility of this method was discussed by reference to the similar tertiary structure prediction of beta-trypsin from the X-ray crystal structure of an elastase.     

Crystal structures of the complexes of a group IIA phospholipase A2 with two natural anti-inflammatory agents, anisic acid, and atropine reveal a similar mode of binding

Secretory low molecular weight phospholipase A(2)s (PLA(2)s) are believed to be involved in the release of arachidonic acid, a precursor for the biosynthesis of pro-inflammatory eicosanoids. Therefore, the specific inhibitors of these enzymes may act as potent anti-inflammatory agents. Similarly, the compounds with known anti-inflammatory properties should act as specific inhibitors. Two plant compounds, (a) anisic acid (4-methoxy benzoic acid) and (b) atropine (8-methyl-8-azabicyclo oct-3-hydroxy-2-phenylpropanoate), have been used in various inflammatory disorders. Both compounds (a) and (b) have been found to inhibit PLA(2) activity having binding constants of 4.5 x 10(-5) M and 2.1 x 10(-8) M, respectively. A group IIA PLA(2) was isolated and purified from the venom of Daboia russelli pulchella (DRP) and its complexes were made with anisic acid and atropine. The crystal structures of the two complexes (i) and (ii) of PLA(2) with compounds (a) and (b) have been determined at 1.3 and 1.2 A resolutions, respectively. The high-quality observed electron densities for the two compounds allowed the accurate determinations of their atomic positions. The structures revealed that these compounds bound to the enzyme at the substrate - binding cleft and their positions were stabilized by networks of hydrogen bonds and hydrophobic interactions. The most characteristic interactions involving Asp 49 and His 48 were clearly observed in both complexes, although the residues that formed hydrophobic interactions with these compounds were not identical because their positions did not exactly superimpose in the large substrate-binding hydrophobic channel. Owing to a relatively small size, the structure of anisic acid did not alter upon binding to PLA(2), while that of atropine changed significantly when compared with its native crystal structure. The conformation of the protein also did not show notable changes upon the bindings of these ligands. The mode of binding of anisic acid to the present group II PLA(2) is almost identical to its binding with bovine pancreatic PLA(2) of group I. On the other hand, the binding of atropine to PLA(2) is similar to that of another plant alkaloid aristolochic acid.     

Heparin reduces the alpha-helical content of cobra basic phospholipase A(2) and promotes its complex formation

The interaction of phospholipase A(2) (PLA(2)) with glycosaminoglycans (GAGs) has recently attracted attention in view of its implication on inflammation and cell proliferation. By using Fourier Transformed Infrared (FTIR) spectroscopic measurements, we demonstrate here that binding of cobra basic phospholipase A(2) from Naja nigricollis (N-PLA(2)) to heparin may induce a significant conformational change observed in the amide I region of the enzyme's alpha-helical and beta-sheet structure. It is observed that notable conformational change of N-PLA(2) due to heparin binding occurs only when heparin's chain length is at least an octasaccharide as evidenced by circular dichroism and optical density measurements. This correlation may be an important factor in the aggregation of N-PLA(2) and N-PLA(2)-heparin complexes. Heparin induced change in conformation of PLA(2) is suggested to be a notable link in understanding the diversity in PLA(2) activity when rendered to the extracellular matrix of cell membranes that is full of GAG molecules.     

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.     

Limitations of phosphatidylcholine/deoxycholate mixtures for the analysis of phospholipase A2 inhibition and activation: illustration with annexins.

The effects of human recombinant lipocortin I (annexin I) and bovine lung calpactin I (annexin II) on porcine pancreatic phospholipase A2 (PLA2) activity in phosphatidylcholine (PC)/deoxycholate (DOC) mixtures were investigated. Annexin-associated decreases in PLA2 activity were observed under some conditions, for example, at high DOC/PC molar ratios; however, activation was observed under other conditions. NaCl, which lowers the non-critical micellar concentration (NCMC) of deoxycholate, caused significant decreases in control PLA2 activity in the absence of annexins, and greater decreases in PLA2 activity in annexin-containing samples, resulting in an apparent increase in inhibition. The PC/DOC substrate mixtures themselves appeared unstable. Despite a large excess of detergent, precipitates were, at times, observed upon incubation of some PC/DOC mixtures at 37 degrees C. Such behavior is of interest in view of the numerous reports of PLA2 inhibition by annexins and annexin-derived peptides in the PC/DOC system. The influence of the annexins on activity in this system is consistent with effects on the phase behavior of the PC/DOC mixture and/or competition with the enzyme for available Ca2+. These results caution against use of the PC/DOC system for analysis of potential PLA2 inhibitors unless the phase behavior of the system is more fully delineated.