Activity Of Pancreatic Phospholipase A2 Research Articles

Effect of oxidation and in vitro intestinal hydrolysis on phospholipid toxicity towards HT29 cell line serving as a model of human intestinal epithelium

Oxidation of food-derived phospholipids (PLs) can influence nutrient digestion and induce oxidative stress in gastrointestinal epithelium. In this study, hen egg yolk PL fraction was used to evaluate the effect of lipoxygenase (LOX)-induced PL oxidation on the rate of PL hydrolysis catalyzed by pancreatic phospholipase A2 (PLA2) in the presence of bile salts (BSs). Then, PL/BS solutions containing native or oxidized PLs were used in in vitro intestinal digestion to assess the effect of PL oxidation and hydrolysis on the toxicity towards HT29 cell line. Based on the obtained results, we suggest that hexanal and (E)-2-nonenal, formed by the decomposition of PL hydroperoxides, inhibited PLA2 activity. The cell exposure to simulated intestinal fluid (SIF) containing BSs decreased HT29 cell viability and significantly damaged cellular DNA. However, the genotoxic effect was reversed in the presence of all tested PL samples, while the protective effect against the BS-induced cytotoxicity was observed for native non-hydrolyzed PLs, but was not clearly visible for other samples. This can result from an overlap of other toxic effects such as lipotoxicity or disturbance of cellular redox homeostasis. Taking into account the data obtained, it was proposed that the PLA2 activity decline in the presence of PL oxidation products may be a kind of protective mechanism against rapid release of oxidized FAs characterized by high cytotoxic effect towards intestinal epithelium cells.

Changes in the Secondary and Tertiary Structures of Secreted Phospholipase A2 upon Activation

Activation of human pancreatic phospholipase A2 (PLA2) in the presence of DPPC/DPPG (7:3) vesicles was induced by a temperature shift from 4 to 38 oC. PLA2 activity was monitored by changes in fluorescence of bis-Pyrene-PC (2.5 mol % in the membranes), while simultaneous far- and near-UV circular dichroism (CD) spectra identified changes in the secondary and tertiary structures of the protein in real time. The 4-to-38 oC temperature shift caused dramatic changes in both bis-Pyrene-PC fluorescence and the protein CD spectra. The monomer fluorescence signal of bis-Pyrene-PC rapidly increased and the excimer signal decreased, demonstrating PLA2 activation. Drastic weakening in the α-helical CD signal of the protein, i.e., a 20% decrease in the n-π* transition intensity at 222 nm, was detected upon enzyme activation. The α-helical signal exhibited a significantly smaller change upon a similar temperature shift under non-catalytic conditions (1 mM EGTA), while little changes were detected in the absence of lipid. Strong changes in the tertiary structure during PLA2 activation were also identified. Initially, at 4 oC, the near-UV CD spectra showed a weak negative band around 280 nm. Upon a shift to 38 oC, strong positive CD bands rapidly developed around 250 and 280 nm, implying significant changes in the conformation and/or the microenvironment of Tyr and Trp side chains of PLA2, possibly accompanied with a global tertiary structure perturbation associated with deformation of the abundant disulfide bonds in the protein. These experiments provide new information on the structure-function relationship of PLA2 by near-simultaneous measurements of PLA2 activity and its secondary and tertiary structures.

Effects of antiflammins on transglutaminase and phospholipase A2 activation by transglutaminase

Two anti-inflammatory peptides, named antiflammins (AFs), corresponding to a region with high amino acid similarity between lipocortin-1 and uteroglobin were tested for their ability to inhibit transglutaminase (TG) and low-molecular-mass phospholipase A2 (PLA2). Porcine pancreatic PLA2 activity and guinea pig hepatic TG activity were determined by arachidonyl release from arachidonyl-phosphatidylcholine and by the incorporation of putrescine into succinylated casein, respectively. AFs inhibited TG activity but did not affect PLA2 activity. Moreover, porcine pancreatic PLA2 was activated by TG and AFs decreased porcine pancreatic PLA2 activation induced by TG. Taken together, our results support the hypothesis that the anti-inflammatory effects of AFs are, at least in part, due to the action of AFs on TG activity.

Bromoacyl Analogues of Phosphatidylcholine with Intramolecular Fluorescence Quenching and Their Use as Substrates for Continuous Monitoring of Phospholipase A2Activity

Two new derivatives of phosphatidylcholine with intramolecular fluorescence quenching were obtained by substituting residues of pyrene butyric and bromine-containing fatty acids for acyl chains. The two compounds can be used for quantitative evaluation of catalytic activity of pancreatic phospholipase A2 in kinetic mode.

Pancreatic phospholipase A2 activity in acute pancreatitis: a prognostic marker for early identification of patients at risk.

Remarkably elevated levels of phospholipase A2 (PLA2) are measurable in human blood samples in cases of acute pancreatitis. The source of the enzyme was first thought to be exclusively the pancreas, but now it is generally accepted that two isoenzymes--the pancreatic PLA2, group I, and the extrapancreatic PLA2, group II--contribute to the raised activity. In contrast to the group II-PLA2, the pancreatic PLA2 is heat-resistant for 1 hour at 60 degrees C. The catalytically inactive proenzyme of the pancreatic PLA2 can be activated by trypsin. The aim of our study was to evaluate the diagnostic value of PLA2 isoenzyme activity measurements to identify patients with severe complications in acute pancreatitis. Blood samples from patients suffering from acute pancreatitis were analyzed for catalytically active pancreatic PLA2 on day 1 and 2 of hospitalization with a modified radiometric Escherichia coli-based PLA2 assay. In 10 of 41 patients clearly elevated values of catalytically active, heat-resistant pancreatic PLA2 (7.2 to 81.2 U/l) were observed. This group of patients was characterized by severe complications (necrotizing pancreatitis, shock, sepsis, respiratory problems) of which two patients subsequently died. Patients with low or undetectable activity (<7 U/l) of pancreatic PLA2 recovered rapidly. According to these results the presence of catalytically active pancreatic PLA2 in serum is associated with severe complications of acute pancreatitis. In contrast to total serum-PLA2, the catalytic concentration of pancreatic PLA2 can serve as a prognostic marker in acute pancreatitis.

Influence of ethanol and phosphatidylethanol on the activity of pancreatic phospholipase A2.

Hydrolysis of dioleoylphosphatidylethanol (DOPEt) and dioleoylphosphatidylcholine (DOPC) catalyzed by phospholipase A2 (PLA2) from porcine pancreas has been studied in single-component and binary liposomes in the absence and in the presence of ethanol. DOPEt (an anionic phospholipid) was found to increase the rate of hydrolysis of zwitterionic DOPC in liposomes under the action of PLA2.

Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme.

Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.

Cationic residues 53 and 56 control the anion-induced interfacial k*cat activation of pancreatic phospholipase A2.

Added NaCl or anionic amphiphiles increase the rate of hydrolysis of dispersions of zwitterionic phospholipid by pancreatic phospholipase A2 (PLA2). Two effects of the negative charge at the interface have been dissected: enhanced binding of the enzyme to the interface, and k*cat activation of the enzyme at the interface [Berg et al. (1997) Biochemistry 36, 14512-14530]. Results reported here show that the structural basis for the k*cat activation is predominantly through cationic K53 and K56 in bovine pancreatic PLA2 with the anionic interface. The maximum rate at saturating diheptanoylphosphatidylcholine micelles, VMapp, for WT, K56M, and K53M in 4 M NaCl is in the 800-1300 s-1 range. In contrast, VMapp at 0.1 M NaCl is considerably higher for K56M (400 s-1) and K53M (230 s-1) compared to the rate with WT (30 s-1) or K56E (45 s-1). The rate of hydrolysis of anionic dimyristoylphosphatidylmethanol vesicles is virtually the same with all these mutants (200-300 s-1) and it is not affected by added NaCl. The chemical step for the hydrolysis of anionic and zwitterionic substrates remains rate-limiting in the presence or absence of added NaCl. A modest (approximately 10-fold) effect of K56M substitution or of added NaCl is seen on the binding of the enzyme to the interface; however, the binding of the substrate or a substrate mimic to the active site of the enzyme at the interface is not affected by more than a factor of 2. Magnitudes of the primary rate and equilibrium parameters at the zwitterionic and anionic interfaces show that the effect of mutation or of added NaCl is primarily on k*cat at the zwitterionic interface. These results are interpreted in terms of a two-state model for the interfacial allosteric activation, where the enzyme-substrate complex at the zwitterionic interface becomes catalytically active only after the positive charge on cationic K56 and K53 has been removed by mutation or neutralized by anionic charges in the interface.

The interaction of phospholipase A2 with liposomes: an immunological approach to its study.

Porcine pancreatic phospholipase A2 (PLA2) has been incubated for 2 hours at 34 degrees C with rabbit anti-pig pancreatic PLA2 polyclonal antiserum in the absence or presence of phosphatidylcholine liposomes in different physical states. Subsequent assay of hydrolysis -triggered through addition of 5 mM Ca2+- at 34 degrees C, shows that preincubation with antiserum in the presence of 1,2-dimyristoyl-3-sn-phosphatidylcholine liquid-crystalline vesicles, renders the PLA2 activity undetectable, similarly to what is found if preincubation is carried out in the absence of liposomes. In contrast, 1,2-dipalmitoyl-3-sn-phosphatidylcholine liposomes, which at 34 degrees C are in the gel-phase, protect the enzyme from the antiserum effect. The results are consistent with a stronger binding of PLA2 to gel phase, as compared to liquid crystalline vesicles and suggest that through the physical interaction with liposomes in the gel state, the enzyme is shielded from reaction with the antibodies. Taking into account the characteristic hydrolysis profiles of vesicles in different physical states, it can be concluded that the above interpretation agrees with the proposal that PLA2-membrane association promotes the interfacial activation of the enzyme.

Zinc (Zn2+) binds to and stimulates the activity of group i but not group II phospholipase A2

Phospholipase A2 plays an important part in the generation of inflammatory lipid mediators and so it is of major interest to understand functional distinctions between structurally similar forms of phospholipase A2. In the present study, the influence of zinc (Zn2+) on the activity of group I and group II phospholipase A2 was examined in vitro. It appeared that Zn2+ (0.04-1 x 10(-3)M) increased group I phospholipase A2 activity from porcine pancreas and rat lung whereas the activity of group II phospholipase A2 from Crotalus atrox and Vipera russelli was unaffected. The presence of Cd2+ of Hg2+ (0.8-5 x 10(-3)M) also increased group I pancreatic phospholipase A2 activity while no augmentation was found with Cr2+, Fe2+ or Mg2+. The selective stimulation of group I phospholipase A2 by Zn2+ corresponded to a binding of these phospholipases A2 to a zinc-affinity column, while group II phospholipase A2 was not bound. Furthermore, the PLA2 activity in bronchoalveolar lavage fluid from rat was stimulated by Zn2+. These results indicate that Zn2+ binds to and increases the activity of group I, but not group II phospholipase A2. This difference in Zn(2+)-binding may be used to discriminate between group I and group II phospholipase A2 and to separate the enzymes from each other in complex biological materials. The possibility that activation of group I phospholipase A2 in the lung is important in zinc-induced metal fume fever is implied.

Pancreaticobiliary maljunction-associated pancreatitis: an experimental study on the activation of pancreatic phospholipase A2.

Congenital dilatation of the bile duct (CDBD), or choledochalcyst, is often complicated by recurrent pancreatitis. Reflux of pancreatic juice into the bile duct through pancreaticobiliary maljunction (PBM), an anomaly commonly associated with CDBD, and ensuing activation of pancreatic enzymes could be involved in the pathophysiologic mechanism of recurrent pancreatitis. A study was undertaken to follow the time course of the activity of phospholipase A2 (PLA2) in animal models of PBM. The assay procedures for PLA2 were evaluated, as were the conditions for separating the active enzyme from its inactive proenzyme (pro-PLA2) by immunoblotting. A rat model was designed according to Block's method with some modifications. The kinetics of prophospholipase A2 (proPLA2) activation in bile was examined by measuring PLA2 activity and by immunoblotting using anti-rat pancreatic enzyme antibody after separating PLA2 from its zymogen under nonreducing conditions. Experimental animals were divided into three groups: group 1 (PBM group) in which bile and pancreatic juice were mixed with occlusion of the papilla; group 2, in which the papilla and hepatic hillus were occluded without mixing the two juices; and group 3, in which simple laparotomy was done. In group 1 animals, pro-PLA2 in bile was activated to its active form. In group 2 animals, where proPLA2 was predominant, there was only slight elevation of PLA2 activity in bile. In group 1 an immunohistologic study demonstrated localization of PLA2 around necrotic foci in the pancreatic parenchyma. These results suggest the involvement of activated PLA2 in the pathogenesis of choledochal cystassociated pancreatitis.

Lipopolysaccharide induced apoptosis of rat pancreatic acinar cells.

Bacterial lipopolysaccharide (LPS) has been proposed to participate in the pathogenesis of pancreatic inflammatory disease. This study investigated the role of endotoxaemia in the pathogenesis of pancreatic acinar cell injury. Sixty eight male Spraque-Dawley rats were used in the study. Escherichia coli LPS (5 mg/kg) was injected into the peritoneal cavity of the rats. The concentration of pancreatic phospholipase A2 (PLA2) in plasma was measured and pancreatic tissue examined by histology, in situ detection of free DNA 3'-ends, and electrophoretic DNA analysis. The concentration of pancreatic PLA2 increased in plasma and the catalytic activity of PLA2 increased in pancreatic tissue after an LPS injection. Apoptosis in pancreatic acinar cells and fragmentation of DNA typical of apoptosis in pancreatic tissue was seen 24 hours after an LPS injection. Pancreatic acinar atrophy was seen 72 hours after the LPS injection. These data show that LPS causes release of pancreatic PLA2 into blood plasma, activation of PLA2 in pancreatic tissue, and apoptosis of acinar cells.

Targeting of Porcine Pancreatic Phospholipase A2 to Human Platelets: Introduction of an RGD Sequence by Genetic Engineering

The possibility to induce specific disruption of activated platelets by binding of porcine pancreatic phospholipase A2 (PLA2) was tested by constructing a set of PLA2-mutants containing an Arg-Gly-Asp (RGD) sequence. One mutant was made with RGD as part of a surface-exposed loop (RGDloop). Four mutants were made with RGD as part of a C-terminal extension: one with RGD directly coupled to the C-terminus (RGDc) and three mutants (CRSx) with x= 22, 42 and 82 hydrophylic non-charged amino acids between RGD and the enzyme. All mutants retained 20-80% activity of native PLA2 and showed little binding to resting platelets. The binding of the native enzyme and RGDloop was not increased following stimulation. In contrast, the mutants RGDc and CRSx showed stimulation-dependent binding to the platelet receptor GPIIb/IIIa, since GRGDS-peptide and a monoclonal antibody against the complex interfered with binding. In alpha-thrombin-stimulated platelets, CRS42 and CRS82 induced about 5% hydrolysis of [3H]-arachidonic acid-labeled phospholipids. Stimulation with a combination of alpha-thrombin and collagen (known to expose phosphatidylserine) increased hydrolysis to 11%. Despite the membrane disruption, the cells did not leak lactate dehydrogenase. We conclude that PLA2 can be targeted to activated platelets by introducing RGD in a C-terminal extension with a minimum distance (42 amino acids) between RGD and the enzyme. However, more hydrolytic activity is required to eliminate activated platelets among a suspension of resting platelets and other blood cells.

Phosphatidylcholine as substrate for human pancreatic phospholipase A2. Importance of the physical state of the substrate.

The long-chain phosphatidylcholine/sodium cholate aqueous system as substrate for human pancreatic phospholipase A2 (PLA2) was investigated. At a constant phosphatidylcholine (PC) concentration of 8 mM, the enzyme activity increased with a decrease in cholate (C) concentration up to a PC/C ratio of approximately 0.8 and then rather abruptly decreased to lower values at a ratio above 1.5. At ratios between 0.8 and 1.5, an increasing lag phase in the PLA2 activity was seen, indicating a progressive decrease in substrate availability to the enzyme. Reaction mixtures with a PC/C ratio of up to 0.67 were optically clear solutions composed of mixed bile salt/PC micelles of increasing mixed micellar aggregate size. Ratios between 0.67 and 1.5 were characterized by an increase in turbidity (at 330 and 450 nm) due to increasing formation of vesicles or liposomes. Above a PC/C ratio of 1.5, a sharp increase in turbidity was seen due to increasing formation of bilayer structures other than vesicles. Pure vesicles obtained by dialysis of mixed micellar solutions were not hydrolyzed by the enzyme. Addition of bile salts reversed the inhibition which was accompanied by a decrease in turbidity. Phosphatidylcholine was preferred as substrate for human PLA2 when present in large mixed disc-like bile salt micelles. Vesicular or other types of lamellar liquid-crystalline phases of long-chain phosphatidylcholine did not serve as substrate for PLA2.

Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities

A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246-254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726-730 [1988]). P2 (0.05 microM) was also reported to inhibit porcine pancreatic phospholipase A2 (PLA2). The effect of synthetic P2 (98% pure) on PLA2 was evaluated in two assay systems. Using porcine pancreatic PLA2 and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005-50 microM) had no effect on PLA2 activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation. In another assay, using human synovial fluid PLA2 as the enzyme and [14C]-oleate-labelled E. coli substrate, P2 (0.005-50 microM) had no significant effect on PLA2 activity. A reported PLA2 inhibitor, manoalide, was a potent inhibitor of PLA2 in both assay systems. On the basis of these results, we conclude that P2 is devoid of PLA2 inhibitory activity.

Bilayer packing stress and defects in mixed dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine and their susceptibility to phospholipase A2

The hydrolysis of mixed dilinoleoylphosphatidylethanolamine (DiLinPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) dispersions by porcine phospholipase A2, under conditions leading to the bilayer-to-nonbilayer phase transition, has been studied. Two structurally distinct forms of the dispersions were used, multilamellar vesicles (MLV) and supercritical large unilamellar vesicles (SCLUV). In MLV, maximum free fatty acid was produced in dispersions containing 85 mol % DiLinPE. The peak in the fatty acid release is found at the onset of appearance of the nonbilayer defects reported earlier. DiLinPE was found to be preferentially hydrolyzed as compared to POPC. When cholesterol was added to the mixed DiLinPE/POPC MLV, the onset of the observable appearance of nonbilayer defects, the positions of the peaks for total hydrolysis, and the preferential hydrolysis of DiLinPE were all shifted toward lower DiLinPE concentrations. In SCLUV, where the appearance of nonbilayer structures is prevented by constraining the lipids in bilayer configuration, the hydrolysis by PLA2 increases with increasing DiLinPE as predicted from the increase in the calculated monolayer bending energy. The results are interpreted to be related to the pretransition molecular-packing stress and defects at the onset of the bilayer-to-nonbilayer transition. Results indicate that the porcine pancreatic phospholipase A2 activity is controlled by bilayer-packing stress, which may cause structural defects of the substrate, among other factors. Results also indicate a preferential localization of PE at stress-related defect regions.

Modulation of phospholipase A2 activity by the tumour promoters phorbol esters and teleocidin

The effects of tumour promoters, namely phorbol esters and teleocidin, on the activity of porcine pancreatic phospholipase A2 (PLA2) was investigated by using a system of small unilamellar vesicles composed of dipalmitoyl-phosphatidylcholine (DPPC). DPPC vesicles encapsulating Quin 2 (Quin 2/DPPC vesicles) were suspended in a medium containing Ca2+. The addition of PLA2 to Quin 2/DPPC vesicles increased the fluorescence intensity of Quin 2. This increase was due to chelation of Quin 2 with Ca2+, which resulted from an increase in the permeability of the phospholipid bilayer caused by the hydrolytic activity of PLA2. The tumour promoters phorbol 12-myristate 13-acetate (PMA) and teleocidin, at low concentrations, enhanced PLA2 activity at temperatures below the phase-transition temperature of the membrane, but, in contrast, high concentrations of the tumour promoters suppressed PLA2 activity. Phorbol 12-myristate (PM) also had a similar effect on PLA2 activity. PMA and PM disturbed the membrane structure markedly, which was indicated by the enhanced leakage of carboxyfluorescein (CF) from DPPC vesicles encapsulating CF. On the other hand, phorbol 12,13-didecanoate and 4 alpha-phorbol 12,13-didecanoate, which did not disturb the membrane structure to the same extent, had an insignificant effect on PLA2 activity. It is therefore concluded that PLA2 catalyses the hydrolysis of phospholipids in bilayer vesicles which contain a moderate degree of structural defects. However, the effects of tumour promoters on PLA2 activity was not related to their potencies as inflammatory and tumour-promoting agents.

The role of phospholipase A2 in human acute pancreatitis.

Several studies suggest that the activation of pancreatic phospholipase A2 (PLA2) and its release from injured acinar cells play an important role in the pathogenesis of acute pancreatitis. Elevated catalytic activity of PLA2 in serum is associated especially with severe forms of the disease. PLA2 has been purified from human cadaver pancreas and an antiserum raised against the enzyme in rabbits. Immuno-histochemical localization of PLA2 in pancreatic tissue was abnormal in acute pancreatitis. A time-resolved fluoroimmunoassay for human pancreatic PLA2 has been developed. Increased serum concentrations of immunoreactive PLA2 were found in acute pancreatitis during the first week after hospital admission. The values returned to normal somewhat more slowly than corresponding serum amylase values. The immunochemical determination of PLA2 in serum provides a fast and specific detection of injury to pancreatic acinar cells. The pancreas is not the only source of PLA2 in acute pancreatitis. The nonpancreatic PLA2 may originate from various inflammatory cells, but this hypothesis remains to be proven.

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.