Regulation Of Phospholipase A2 Activity Research Articles

Characterization and proteomic analysis of plasma-derived small extracellular vesicles in locally advanced rectal cancer patients.

Neoadjuvant chemoradiotherapy (nCRT) stands as a pivotal therapeutic approach for locally advanced rectal cancer (LARC), yet the absence of a reliable biomarker to forecast its efficacy remains a challenge. Thus, this study aimed to assess whether the proteomic compositions of small extracellular vesicles (sEVs) might offer predictive insights into nCRT response among patients with LARC, while also delving into the proteomic alterations within sEVs post nCRT. Plasma samples were obtained from LARC patients both pre- and post-nCRT. Plasma-derived sEVs were isolated utilizing the TIO2-based method, followed by LC-MS/MS-based proteomic analysis. Subsequently, pathway enrichment analysis was performed to the Differentially Expressed Proteins (DEPs). Additionally, ROC curves were generated to evaluate the predictive potential of sEV proteins in determining nCRT response. Public databases were interrogated to identify sEV protein-associated genes that are correlated with the response to nCRT in LARC. A total of 16 patients were enrolled. Among them, 8 patients achieved a pathological complete response (good responders, GR), while the remaining 8 did not achieve a complete response (poor responders, PR). Our analysis of pretreatment plasma-derived sEVs revealed 67 significantly up-regulated DEPs and 9 significantly down-regulated DEPs. Notably, PROC (AUC: 0.922), F7 (AUC: 0.953) and AZU1 (AUC: 0.906) demonstrated high AUC values and significant differences (P value < 0.05) in discriminating between GR and PR patients. Furthermore, a signature consisting of 5 sEV protein-associated genes (S100A6, ENO1, MIF, PRDX6 and MYL6) was capable of predicting the response to nCRT, yielding an AUC of 0.621(95% CI: 0.454-0.788). Besides, this 5-sEV protein-associated gene signature enabled stratification of patients into low- and high-risk group, with the low-risk group demonstrating a longer overall survival in the testing set (P = 0.048). Moreover, our investigation identified 11 significantly up-regulated DEPs and 31 significantly down-regulated DEPs when comparing pre- and post-nCRT proteomic profiles. GO analysis unveiled enrichment in the regulation of phospholipase A2 activity. Differential expression of sEV proteins distinguishes between GR and PR patients and holds promise as predictive markers for nCRT response and prognosis in patients with LARC. Furthermore, our findings highlight substantial alterations in sEV protein composition following nCRT.

Novel and rare functional genomic variants in multiple autoimmune syndrome and Sjögren's syndrome.

BackgroundMultiple autoimmune syndrome (MAS), an extreme phenotype of autoimmune disorders, is a very well suited trait to tackle genomic variants of these conditions. Whole exome sequencing (WES) is a widely used strategy for detection of protein coding and splicing variants associated with inherited diseases.MethodsThe DNA of eight patients affected by MAS [all of whom presenting with Sjögren’s syndrome (SS)], four patients affected by SS alone and 38 unaffected individuals, were subject to WES. Filters to identify novel and rare functional (pathogenic–deleterious) homozygous and/or compound heterozygous variants in these patients and controls were applied. Bioinformatics tools such as the Human gene connectome as well as pathway and network analysis were applied to test overrepresentation of genes harbouring these variants in critical pathways and networks involved in autoimmunity.ResultsEleven novel and rare functional variants were identified in cases but not in controls, harboured in: MACF1, KIAA0754, DUSP12, ICA1, CELA1, LRP1/STAT6, GRIN3B, ANKLE1, TMEM161A, and FKRP. These were subsequently subject to network analysis and their functional relatedness to genes already associated with autoimmunity was evaluated. Notably, the LRP1/STAT6 novel mutation was homozygous in one MAS affected patient and heterozygous in another. LRP1/STAT6 disclosed the strongest plausibility for autoimmunity. LRP1/STAT6 are involved in extracellular and intracellular anti-inflammatory pathways that play key roles in maintaining the homeostasis of the immune system. Further; networks, pathways, and interaction analyses showed that LRP1 is functionally related to the HLA-B and IL10 genes and it has a substantial impact within immunological pathways and/or reaction to bacterial and other foreign proteins (phagocytosis, regulation of phospholipase A2 activity, negative regulation of apoptosis and response to lipopolysaccharides). Further, ICA1 and STAT6 were also closely related to AIRE and IRF5, two very well known autoimmunity genes.ConclusionsNovel and rare exonic mutations that may account for autoimmunity were identified. Among those, the LRP1/STAT6 novel mutation has the strongest case for being categorised as potentially causative of MAS given the presence of intriguing patterns of functional interaction with other major genes shaping autoimmunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0525-x) contains supplementary material, which is available to authorized users.

A genome‐wide survey of copy number variation regions in various chicken breeds by array comparative genomic hybridization method

The discovery of copy number variation (CNV) in the genome has provided new insight into genomic polymorphism. Studies with chickens have identified a number of large CNV segments using a 385k comparative genomic hybridization (CGH) chip (mean length >140 kb). We present a detailed CNV map for local Chinese chicken breeds and commercial chicken lines using an Agilent 400k array CGH platform with custom-designed probes. We identified a total of 130 copy number variation regions (CNVRs; mean length = 25.70 kb). Of these, 104 (80.0%) were novel segments reported for the first time in chickens. Among the 104 novel CNVRs, 56 (53.8%) of the segments were non-coding sequences, 65 (62.5%) showed the gain of DNA and 40 (38.5%) showed the loss of DNA (one locus showed both loss and gain). Overlapping with the formal selective sweep data and the quantitative trait loci data, we identified four loci that might be considered to be high-confidence selective segments that arose during the domestication of chickens. Compared with the CNVRs reported previously, genes for the positive regulation of phospholipase A2 activity were discovered to be significantly over-represented in the novel CNVRs reported here by gene ontology analysis. Availability of our results should facilitate further research in the study of the genetic variability in chicken breeds.

Regulation of phospholipase A2 activity in undifferentiated and neutrophil-like HL60 cells. Linkage between impaired responses to agonists and absence of protein kinase C-dependent phosphorylation of cytosolic phospholipase A2.

We compared the regulation of cytosolic phospholipase A2 (cPLA2) activity in undifferentiated and neutrophil-like HL60 cells. Although Ca(2+)-mobilizing P2-purinergic receptors are expressed in both cell types, arachidonic acid (AA) release stimulated by P2-purinergic agonists was 5-7-fold higher in the differentiated cells. Similarly, the stimulation of AA release by AlF4- in intact cells or by ATP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in electropermeabilized cells was significantly higher in the differentiated cells. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced A23187-stimulated AA release in intact HL60 granulocytes with minimal effects in the undifferentiated cells. Immunoblotting experiments showed similar levels of cPLA2 and of agonist-mediated activation of mitogen-activated protein kinase in both cell types. Experiments measuring stimulation of AA release by either melittin, using endogenously labeled intact cells, or Ca2+, using homogenates and exogenous substrate, indicated that undifferentiated cells do not lack an activatable PLA2. The stimulatory effects of GTP gamma S and Ca2+ on AA release in homogenates from endogenously labeled cells suggested that undifferentiated cells display G protein-cPLA2 coupling. Basal and PMA-stimulated phosphorylation of cPLA2 was detected in differentiated, but not in undifferentiated cells. However, the two cell types displayed only subtle differences in the time courses of phosphorylation of mitogen-activated protein kinase triggered by agonists and PMA. The observed defect in cPLA2 phosphorylation may represent the alteration preventing agonist-mediated stimulation of AA release in undifferentiated HL60 cells.

Effects of endotoxin and dexamethasone on group I and II phospholipase A2 in rat ileum and stomach.

Phospholipase A2 (EC 3.1.1.4) is a key enzyme in inflammation and is thought to play an important part in inflammatory diseases of the gastrointestinal tract. To investigate the nature and regulation of phospholipase A2 activity in the gastrointestinal mucosa, the distribution of messenger ribonucleic acid (mRNA) for group II phospholipase A2 in various parts of the rat gastrointestinal tract was studied, as well as the influence of endotoxin or dexamethasone, or both, on the group I and II phospholipase A2 mRNA expression and activity in the rat glandular stomach and distal ileum. The results show that (a) group II phospholipase A2 is present along the whole gastrointestinal tract, but in particularly large amounts in the distal ileum, (b) endotoxin increases group II, but not group I, phospholipase A2 mRNA expression in the glandular stomach and distal ileum, and (c) dexamethasone reduces the endotoxin induced increases in group II phospholipase mRNA expression and activity in the gastrointestinal mucosa. These findings suggest that phospholipase A2 of type II is a mediator of endotoxin effects in the gastrointestinal mucosa and that its expression at the mRNA level can be inhibited by corticosteroids.

Inhibition of Human Skin Phospholipase A2 by “Lipocortins” Is an Indirect Effect of Substrate/Lipocortin Interaction

Proteins of the annexin/lipocortin family have been claimed to mediate the anti-inflammatory action of glucocorticosteroids by the inhibition of phospholipases A2. This hypothesis has been challenged by the finding that annexins do not directly interact with the enzyme in a classical enzyme/inhibitor behavior, but more likely block the access of the phospholipase A2 to its substrate by binding to phospholipids. Because former studies with skin phospholipase A2 suggested a specific regulation by annexin-1, we investigated the substrate dependence of this effect. For this purpose phospholipase A2 activities in human epidermis and dermis homogenates were measured in the presence of various amounts of annexins-1, -2, or -5. The respective annexin was preincubated in separate series either with the substrate or with the enzyme. We found a partial inhibition of both epidermal and dermal phospholipase A2 activities with all annexins tested (annexin-5 >> annexin-2 > annexin-1). The inhibitory effect was absolutely dependent on the annexin/phospholipid ratio and occurred only at very high annexin concentrations relative to the amount of substrate. Our data demonstrate that the inhibition of human skin phospholipase A2 by annexins depends on the substrate concentrations, as has been shown for phospholipases A2 of other origins as well. All observations can be explained by the current "substrate depletion model" characterizing the indirect effects of annexins on phospholipase A2 activities. It is therefore rather unlikely that annexins are directly involved in the regulation of phospholipase A2 activity of human skin under physiologic conditions.

Influence of isoproterenol and phosphodiesterase inhibitors on platelet-activating factor biosynthesis in the human neutrophil.

Increasing cellular levels of cAMP inhibit eicosanoid production in the human neutrophil; however, little is known about the effects of cAMP on platelet-activating factor (PAF) biosynthesis in this cell. In the current study, the beta-adrenergic receptor agonist isoproterenol, alone or in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), was used to increase cAMP in neutrophils. The incorporation of [3H]acetate into PAF and the synthesis of leukotrienes in response to ionophore A23187 were significantly inhibited by 10 microM isoproterenol. The inhibitory effect on PAF was potentiated by the addition of 10 microM IBMX. The effects of IBMX were mimicked by rolipram, an inhibitor of the cAMP-specific phosphodiesterase IV. Mass spectrometric analysis of the PAF molecular species in stimulated neutrophils indicated that the combination of isoproterenol and IBMX inhibited (> 50%) ionophore- and fMLP-induced production of PAF. To better understand the mechanism involved in the inhibition of PAF formation, the major biosynthetic steps were examined in the presence and absence of a maximally effective concentration of isoproterenol and IBMX. Isoproterenol alone or in the presence of IBMX had no measurable effect on the ionomycin-induced increase in cytosolic calcium concentration, as assessed by fura-2 fluorescence. Treating intact neutrophils with a combination of isoproterenol and IBMX did not inhibit acetyltransferase activity when assayed in a subsequent broken cell preparation. Finally, increasing cellular cAMP with these drugs did not influence the ability of the neutrophil to catabolize PAF. Phospholipase A2-like activity was assayed in the whole cell by measuring the mobilization of phospholipase A2 products, PAF, lyso PAF, and arachidonic acid, from cellular phosphoglycerides. Treatment of neutrophils with isoproterenol and IBMX significantly reduced the production of lyso PAF and PAF from 1-alkyl-2-arachidonoyl sn-glycero-3-phosphocholine. Similarly, increasing cellular levels of cAMP inhibited the cell's ability to mobilize arachidonic acid upon cell activation. These data suggest that increasing cellular levels of cAMP leads to the inhibition of PAF and leukotriene biosynthesis, at least in part, by regulation of phospholipase A2 activity.

IL-1 increases phospholipase A2 activity, expression of phospholipase A2-activating protein, and release of linoleic acid from the murine T helper cell line EL-4.

The early events in IL-1-mediated activation of T cells were investigated in the murine T cell line, EL-4. Treatment of EL-4 cells with human rIL-1 beta resulted in a rapid increase in phospholipase A2 (PLA2) activity. PLA2 activity increased approximately fivefold within 4 min after exposure to IL-1. Synthesis of the phospholipase A2- activating protein (PLAP) and its mRNA were also increased within 4 min of IL-1 treatment and preceded the increase in PLA2 enzyme activity. The increases in PLA2 activity and PLAP protein and mRNA levels were all transient and declined to baseline within 10 min after the addition of IL-1. The changes in levels of PLAP as a function of time after IL-1 treatment were consistent with PLAP playing an important role in the regulation of PLA2 activity in this system. The consequence of the elevated PLA2 activity was examined by analysis of the fatty acids released from IL-1-treated cells. There was a 20-fold increase in the release of radioactivity from [14C]-linoleic acid labeled cells whereas there was very little change in the release of radioactivity from [14C]-arachidonic acid labeled cells in response to the addition of IL-1. The radioactivity released from [14C]-linoleic acid labeled cells was analyzed by HPLC; no conversion of radiolabeled linoleic into arachidonic acid was observed. In EL-4 cells, IL-1 potentiates PMA-mediated release of IL-2 at suboptimal concentrations of PMA. Linoleic acid also augmented PMA-induced IL-2 release from the EL-4 cells. This fatty acid was more than 10 times more effective than arachidonic acid in this regard. Furthermore, the addition of exogenous PLAP to EL-4 cells could substitute for IL-1 in the stimulation of IL-2 release. These results suggest that the IL-1 effects on T cells may be mediated at least in part through increased PLA2 activity due to increased synthesis of PLAP. Furthermore, the release of the unsaturated fatty acid linoleic acid or its metabolites may be of functional importance in IL-1-mediated IL-2 production by EL-4 cells.

Arachidonic acid release by basophilic leukemia cells and macrophages stimulated by Ca2+ ionophores, antigen and diacylglycerol: essential role for protein kinase C and prevention by glucocorticosteroids

The role of protein kinase C in phospholipase A2 (PLA2) activation in rat basophilic leukemia cells (RBL-2H3) and macrophages was investigated. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) doubled ionomycin-induced PLA2 activity, assessed by [3H]arachidonate release. Protein kinase C inhibitors, staurosporine and K252a (100 nM) or H-7 (15 μg/ml) inhibited ionomycin-stimulation of PLA2 activity by 62, 75 and 80%, respectively. Down-regulation of protein kinase C by prolonged treatment with TPA inhibited Ca2+-ionophore A23187 or antigen-stimulation of [3H]arachidonate release by 80%. We examined whether the inhibitory effect of dexamethasone (DEX) on PLA2 activity is related to modulation of protein kinase C activity. The 50% inhibition by DEX of ionomycin elevation of [3H]arachidonate release was almost overcome by addition of TPA. The Ca2+ ionophore and antigen-induced increase in [3H]TPA binding to intact RBL cells was not impaired by DEX. However, DEX markedly reduced phosphorylation of several proteins. 1-Oleoyl-2-acetyl-glycerol (OAG) had a sustained stimulatory effect on PLA2 activity in isolated plasma membranes derived from treated bone-marrow intact mouse macrophages, while both DEX and staurosporine reduced elevated PLA2 activity by 68 and 84%, respectively. The results support an essential role for protein kinase C in regulation of PLA2 activity.

A G Protein Mutant That Inhibits Thrombin and Purinergic Receptor Activation of Phospholipase A 2

The stimulation of phospholipase A2 by thrombin and type 2 (P2)-purinergic receptor agonists in Chinese hamster ovary cells is mediated by the G protein Gi. To delineate alpha chain regulatory regions responsible for control of phospholipase A2, chimeric cDNAs were constructed in which different lengths of the alpha subunit of Gs (alpha s) were replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). When a carboxyl-terminal chimera alpha s-i(38), which has the last 38 amino acids of alpha s substituted with the last 36 residues of alpha i2, was expressed in Chinese hamster ovary cells, the receptor-stimulated phospholipase A2 activity was inhibited, although the chimera could still activate adenylyl cyclase. Thus, alpha s-i(38) is an active alpha s, but also a dominant negative alpha i molecule, indicating that the last 36 amino acids of alpha i2 are a critical domain for G protein regulation of phospholipase A2 activity.

Role of protein kinase C in the regulation of phospholipase A2 activity in human platelets

Role of protein kinase C in the regulation of phospholipase A2 activity in human platelets

CD2 triggering stimulates a phospholipase A2 activity beside the phospholipase C pathway in human T lymphocytes.

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.

Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes

G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.

Activation of alpha 1-adrenoceptors, protein kinase C, or treatment with intracellular free Ca2+ elevating agents increases pineal phospholipase A2 activity. Evidence that protein kinase C may participate in Ca2+-dependent alpha 1-adrenergic stimulation of pineal phospholipase A2 activity.

The regulation of pineal phospholipase A2 activity was studied indirectly by measuring the release of [3H]arachidonic acid from [3H]arachidonic acid-labeled tissue in organ culture and the formation of radiolabeled lysophosphatidylcholine by glands labeled with 32Pi or [14C]choline. Glands were transferred sequentially through a series of 10-min incubations in label-free medium. Norepinephrine (10(-5) M) stimulated [3H]arachidonic acid release by 2-fold; release peaked during the first 10 min and returned to basal levels during the third incubation period. Studies with selective alpha 1-, alpha 2-, and beta-adrenergic agents indicated that norepinephrine was acting through alpha 1-adrenergic receptors. Ca2+ appears to play a critical role because the effects of norepinephrine were mimicked by treatment with the Ca2+ ionophore A23187 and inhibited by inorganic Ca2+ channel blockers or EGTA; other [Ca2+]i elevating treatments also stimulated [3H]arachidonic acid release. The possibility that protein kinase C may be involved was studied because it is activated by the alpha 1-adrenergic agonist phenylephrine in the pineal gland (Sugden, D., Vanecek, J., Klein, D. C., Thomas, T. P., and Anderson, W. B. (1985) Nature 314, 359-361). Three protein kinase C activators stimulated [3H]arachidonic acid release with the same relative potency as that established for activation of protein kinase C (4 beta-phorbol 12-myristate 13-acetate greater than 4 beta-phorbol 12,13-dibutyrate greater than 1-oleoyl 2-acetylglycerol). The effects of norepinephrine, A23187, and protein kinase C activators appear to be mediated by phospholipase A2 because the effects of these compounds on [3H]arachidonic acid release are blocked by an established inhibitor of this enzyme, mepacrine, and because these compounds stimulate the formation of 32P- and 14C-labeled lysophosphatidylcholine by glands incubated with 32Pi or [14C]choline. In addition, an inhibitor of diacylglycerol lipase, another enzyme which generates arachidonic acid, did not inhibit the stimulation of [3H]arachidonic acid release by norepinephrine, A23187, or a phorbol ester. Cyclic nucleotides do not appear to play an important role in the regulation of phospholipase A2 activity because dibutyryl cyclic AMP does not alter [3H]arachidonic acid release and also because the amounts of cAMP and cGMP in the culture medium are not consistently associated with [3H]arachidonic acid release. These findings suggest that pineal phospholipase A2 activity is controlled by norepinephrine acting via an alpha 1-adrenergic mechanism which might involve Ca2+ and protein kinase C.

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.

Regulation of phospholipase A2 activity by different lipid-water interfaces.

The activity of pancreatic phospholipase A2 (EC 3.1.1.4) is controlled not only by the architecture of the catalytic site, but is also strongly dependent on the penetrating power of the interface recognition site and the packing density of the lipid-water interface. The influence of the latter two factors on the interface activity has been investigated using chemically modified phospholipases A2 in which the NH2-terminal L-Ala8 has been replaced by DL-[3-13C]Ala, or in which the polypeptide chain has been elongated with DL-[3-13C]Ala. The [DL-(3-13C)Ala8]phospholipase A2 could be resolved into the pure diastereoisomers, [D-(3-13C)Ala8]phospholipase A2 and [L-(3-13C)Ala8]phospholipase A2 by elution on Sephadex G-100 in the presence of a micellar lipid-water interface, as well as by conventional ion exchange chromatography on carboxymethylcellulose. Similar procedures did not effect, however, a separation of DL-[3-13C]Ala7-phospholipase A2 into their respective diasteroisomers, indicating the strategic role of the NH2-terminal L-Ala8 residue in the interaction process between the enzyme and lipid-water interfaces. Kinetic experiments using various micellar short chain lecithins revealed the apparent absence of an interface recognition site in [D-(3-13C)Ala8]- and DL-[3-13C]Ala7-phospholipase A2, while these proteins still possess considerable enzymatic activity toward monomeric substrates. In contrast, however, kinetic experiments using monomolecular surface films, allowing a continuous change in surface density of the substrate molecules, revealed that [D-(3-13C)Ala8]- and DL-[3-13C]Ala7-phospholipase A2 at low surface pressure possess about 60 and 30% of the interface activity of native phospholipase A2, respectively. These results therefore suggest that the modified phospholipases A2 do possess an interface recognition site although less powerful as compared to that of the native enzyme, enabling the estimation of the surface density of micellar short chain lecithins.