Serine Esterase Inhibitor Research Articles

Neuropathy target esterase (NTE): molecular characterisation and cellular localisation.

Organophosphorous esters (OP) are covalent inhibitors of serine esterases. Those OPs which inhibit acetylcholinesterases and are thus acutely toxic have been used worldwide as insecticides. Different physicochemical properties of the tri-aryl phosphates have lead to their widespread use as plasticizers and flame-retardants. Certain OPs, for instance some saligenin cyclic phosphates which are metabolites of tri-aryl phosphates, have relatively little acute anti-cholinesterase directed toxicity, but cause a paralytic neuropathy which is manifest 1–3 weeks after exposure. Two major epidemics of human OP-induced neuropathy have occurred in the past 60 years.

Neuropathy target esterase (NTE): Molecular characterisation and cellular localisation

Organophosphorous esters (OP) are covalent inhibitors of serine esterases. Those OPs which inhibit acetylcholinesterases and are thus acutely toxic have been used worldwide as insecticides. Different physicochemical properties of the tri-aryl phosphates have lead to their widespread use as plasticizers and flame-retardants. Certain OPs, for instance some saligenin cyclic phosphates which are metabolites of tri-aryl phosphates, have relatively little acute anti-cholinesterase directed toxicity, but cause a paralytic neuropathy which is manifest 1–3 weeks after exposure. Two major epidemics of human OP-induced neuropathy have occurred in the past 60 years.

Acyl-Coenzyme A:Cholesterol O -Acyltransferase Is Not Identical to Liver Microsomal Carboxylesterase

Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, DL-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein.

Some studies on the effects of α-chymotrypsin on mast cells from the rat and other species

We have examined the effect of α-chymotrypsin on isolated mast cells from different sources. The enzyme induced a dose-dependent secretion of histamine from purified and non-purified populations of rat peritoneal mast cells. The release was non-cytotoxic and was inhibited by metabolic blockers and extremes of temperature. The process was relatively slow, being essentially complete within 20 min, and was unaffected by phosphatidylserine. A substantial component of the secretion persisted in the absence of extracellular Ca 2+. The release was suppressed by extremes of pH and a variety of anti-allergic compounds and serine esterase inhibitors. In addition to the secretion of preformed mediators, α-chymotrypsin also induced the metabolism of arachidonic acid, resulting in the release of prostaglandin D 2 in a dose-related manner from purified rat peritoneal mast cells. α-Chymotrypsin exhibited a marked tissue and species selectivity in its action and tissue mast cells of the rat, guinea pig and human were generally resistant to the enzyme except at cytotoxic concentrations. On the basis of these results, the possible role of endogenous serine esterases in mast cell activation is discussed.

Cytolytic effector mechanisms of human CD4 + cytotoxic T lymphocytes

To elucidate mechanisms by which human CD4 + cells mediated cytolytic activity, we studied the expression of cytolytic proteins and the effects of inhibitors and mAbs on T-cell clones. Of seven cytolytic CD4 + clones, three were specific for the HLA-DR17, while four recognized DR18. Anti-HLA-DR mAb and anti-CD4 mAb blocked lysis. In addition, N α - p-tosyl- l-lysine chloromethylketone (TLCK), a serine esterase inhibitor, as well as cytochalasin B and monensin, antagonists of secretory pathways, inhibited CD4 + CTLs, whereas the absence of extracellular Ca ++ or the presence of Ca ++ channel blockers partially inhibited cytotoxicity. CD4 + CTLs induced apoptosis of target cell nuclei and membrane damage simultaneously. The CD4 + clones synthesized petforin and granzyme B and expressed the granule-associated protein TIA-1. Our studies indicate that two distinct mechanisms may contribute to cytolysis by CD4 + clones: (1) a Ca ++-dependent mechanism associated with the cytotoxic granules and (2) a Ca ++-insensitive mechanism.

Purification, properties, and specificity of rat brain cytosolic fatty acyl coenzyme A hydrolase.

Rat brain cytosolic acyl-CoA hydrolase has been purified 3,500-fold to apparent homogeneity using heat treatment, ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and hydroxyapatite c chromatography. The purified enzyme remains stable only in the presence of a high concentration (30%, vol/vol) of ethylene glycol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme shows a single band of 40.9 kDa. However, on high-performance size-exclusion chromatography the migration rate of the enzyme corresponds with an apparent molecular mass of 148 kDa, indicating that the native enzyme may be a tetramer. The enzyme catalyzes the hydrolysis of fatty acyl-CoAs from six to 18 carbon chains long, having the highest activity for lauroyl (12:0)-CoA. For the purified enzyme the Km for palmitoyl-CoA is 5.8 microM and the Vmax is 1,300 mumol/min/mg of protein. The enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carnitine. Among the sulfhydryl agents, only p-hydroxymercuribenzoate inhibited the enzyme. The enzyme is also inactivated by treatment with a high concentration of diethyl pyrocarbonate, an active center histidine-reacting agent, but not by phenylmethylsulfonyl fluoride (10 mM), a serine esterase inhibitor. The purified enzyme does not appear to possess any O-ester hydrolase, lysophospholipase, transacylase, or acyltransferase activity.

Simultaneous initiation of degranulation and inhibition of leukotriene release by soman in human basophils

Previous studies noted that the serine esterase inhibitor, soman, could induce histamine release from human basophils. To investigate the mechanisms by which soman causes histamine release (a preformed mediator), we also examined its ability to induce leukotriene release (a newly synthesized mediator) from basophils. We found that no leukotriene release followed activation with soman, while histamine release was usually greater than 70%. In addition, soman and diisopropyl-fluorophosphate were found actively to suppress low level spontaneous leukotriene release as well as ongoing leukotriene release induced by anti-IgE antibody. Soman (0.3 mM) was able to stop leukotriene release as rapidly as the calcium chelator, EDTA. In a series of control experiments, it was noted that soman did not influence the metabolism of LTC 4 to LTD 4 or LTE 4 (for which little metabolism occurred), eliminating the possibility that reduced LTC 4 release could have resulted from its enhanced metabolism. Therefore, using one compound (soman), basophils could be simultaneously activated to degranulate while having the pathway leading to leukotriene release actively suppressed. These results provide further evidence that histamine and leukotriene release are independent pathways resulting from the activation of basophils.

Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase

We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases.

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells.

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 microM of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A2 activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfluoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A2 activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A2 activities and arachidonic acid release in cells pretreated with nominal Ca2+ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A2 activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A2 activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated A2 activity (ii) in addition to the presence of extracellular Ca2+, release of Ca2+ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A2 activities does not appear to require new RNA or protein synthesis.

Purification of an acyl-CoA hydrolase from rat intestinal microsomes. A candidate acyl-enzyme intermediate in glycerolipid acylation.

We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol suggesting the presence of a cysteine residue(s) at or near the active site. Common serine-esterase inhibitors (NaF, phenylmethylsulfonyl fluoride) and activators (Mg2+, Ca2+) had no effect on the hydrolase activity. The enzyme hydrolyzed (transferred to water) 14-20 carbon acyl-CoA with similar efficiencies and did not utilize glycerophospholipids or mono- and diacylglycerols as potential acyl donors/acceptors. Phospholipids and mono- and diradylglycerols at concentrations below 100 microM or polyclonal antibodies raised against the purified hydrolase did not inhibit the enzyme activity. However, the acyl-CoA hydrolase activity could be immunoprecipitated from solubilized microsomes or purified enzyme preparations with corresponding decrease of the hydrolase activity in the supernatant of the immunoprecipitate. Immunoblotting studies show cross-reactivity with a protein of an identical molecular mass in other rat or human tissues. It is concluded that the microsomal acyl-CoA hydrolase deserves consideration as a candidate acyl-enzyme intermediate in glycerolipid synthesis when associated with appropriate acyltransferases.

A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood

A novel procedure has been developed for the quantitation by flow cytometry of function-associated antigens on neutrophils and monocytes in unlysed, unfixed, peripheral blood samples. Freshly drawn blood anticoagulated with the serine esterase inhibitor, phenylmethylsulphonyl fluoride, is mixed with the vital nucleic acid stain, LDS-751, labelled with monoclonal antibodies for 5 min at 4°C, dilluted and analysed in a five-parameter flow cytometer. The three major leucocyte subpopulations (neutrophils, lymphocytes and monocytes) can be resolved in real time on the basis of their side light scattering and staining intensity with LDS-751 in the FL3 channel (erythrocytes and platelets stain very weakly), whilst the fluorescence intensity due to bound fluorescein isothiocyanate- or phycoerythrin-labelled antibody is monitored simultaneously in the FL1 or FL2 channels respectively. This procedure avoids potential artefacts that can occur due to the use of fixatives, erythrocyte lysing agents, or anticoagulants which are also divalent metal ion chelators. It should be widely applicable for the quantitation of those function-associated antigens, such as adhesion molecules and immune complex receptors, whose surface expression can be rapidly upregulated following activation, as well as for the quantitation of those leucocyte surface antigens whose expression is not subject to rapid modulation.

Role of a membrane-associated serine esterase in the oxidant activation of phospholipase A2 by t-butyl hydroperoxide

Exposure of bovine pulmonary-arterial endothelial cells to the oxidant lipid t-butyl hydroperoxide (t-Bu-OOH) increases cell-membrane-associated phospholipase A2 (PLA2) activity and stimulates arachidonic acid (AA) release. To test the hypothesis that a membrane-associated serine esterase plays an important role in activating PLA2, the present study was undertaken. In addition to increasing PLA2 activity and AA release, t-Bu-OOH also enhances the activity of a membrane-associated serine esterase that cleaves the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester (TAME). Changes in the activity of this membrane-bound serine esterase correlate directly with changes in the activity of PLA2. Serine esterase inhibitors such as phenylmethanesulphonyl fluoride, di-isopropyl fluorophosphate and alpha 1-proteinase inhibitor, and TAME, a synthetic substrate for serine esterase, prevent the increase in serine esterase activity, PLA2 activity and AA release caused by t-Bu-OOH. Pretreatment of the endothelial cells with the antioxidant vitamin E prevents t-Bu-OOH-induced stimulation of AA release and the cell-membrane-associated serine esterase and PLA2 activities. Adding t-Bu-OOH or the serine esterase trypsin to the endothelial-cell membrane fraction also significantly augments PLA2 activity, implying that these treatments activate latent PLA2. These results suggest that t-Bu-OOH stimulates a membrane-associated serine esterase that plays a crucial role in activating PLA2 and releasing AA.

Role of serine esterase in A23187-mediated activation of phospholipase A2 in pulmonary endothelium.

To test the hypothesis that an endothelial cell membrane-associated serine esterase is involved in regulating phospholipase A2 (PLA2), we studied the effect of the calcium ionophore A23187 on intracellular PLA2 activity and arachidonic acid (AA) release in bovine pulmonary arterial endothelial cells. Exposure of these cells to A23187 causes a concentration-dependent increase in PLA2 activity and [14C]AA release. In addition to increasing PLA2 activity and AA release, A23187 enhances the activity of endothelial cell membrane-associated serine esterase that acts on the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester. Serine esterase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, prevent the A23187-mediated increase in serine esterase activity, PLA2 activity, and AA release. Pretreatment of the cells with actinomycin D or cycloheximide does not prevent the A23187-mediated increase in AA release, serine esterase activity, or PLA2 activity. The membrane-associated serine esterase activity directly correlates with membrane PLA2 activity. These results suggest that a membrane-associated serine esterase plays a pivotal role in regulating PLA2 activity after exposure to A23187.

Hydrolysis of myelin basic protein in myelin membranes by granzymes of large granular lymphocytes.

Immune-mediated demyelination initially manifests as a separation of myelin lamellae followed by loss of myelin proteins and eventual loss of myelin membranes. Myelin basic protein, one of the major structural proteins of myelin, is highly vulnerable to various proteases derived from diverse cell types. Killer lymphocytes such as CTL, in addition to other immune effectors, have been implicated in inflammatory demyelination. In addition to a pore forming peptide, the granules of CTL and large granular lymphocytes (LGL) contain a number of serine esterases collectively called as granzymes. We studied the effect of these granule enzymes on myelin and found that LGL granule-extracts (LGL-g) hydrolyzed MBP in myelin membranes. LGL-g also cleaved purified MBP at neutral pH in a Ca2+ independent manner and this hydrolysis was inhibited by serine esterase inhibitors. In addition, absorption of LGL-g with antigranzyme A significantly reduced MBP hydrolysis. These findings implicated the granzymes in LGL-g, especially granzyme A, as the agent causing MBP degradation. Inasmuch as activation of CTL in the vicinity of myelinated axons can lead to granule exocytosis, hydrolysis of MBP by granzymes may be a significant event in myelin destruction.

Adhesion Pad Formation and the Involvement of Cutinase and Esterases in the Attachment of Uredospores to the Host Cuticle.

We have investigated the basis of adhesion of uredospores of the obligately parasitic rust fungus Uromyces viciae-fabae to leaves of its broad bean host. Upon contact with an aqueous environment, spores form a structure that we have termed an adhesion pad. The adhesion pad is formed by both living and autoclaved spores, but only adhesion pads formed by living spores adhered to the cuticle of leaves of the host plant. Treatment of living spores with the serine-esterase inhibitor diisopropyl fluorophosphate prevented the adhesion of the pad to the leaf surface, suggesting a functional role for esterase or cutinase in the process of adhesion. A cutinase and two nonspecific serine-esterases were found to be localized on the surface of spores. These enzymes were released rapidly from the spore surface upon contact with an aqueous environment. The addition of the cutinase and the nonspecific esterases to autoclaved spores restored their ability to adhere to the host cuticle. Thus, whereas pad formation appears to be a passive response to the aqueous environment, the actual adhesion of pads to the host cuticle appears to depend on the cutinase and esterases associated with the spore surface. These results suggest a new role for cutinases and serine-esterases in the fungal infection process.

Preparation of the infection court by Erysiphe graminis f. sp. hordei: cutinase is a component of the conidial exudate

Conidia of Erysiphe graminis f. sp. hordei release a liquid upon contact with a substratum, either the surface of a barley leaf or cellophane. The liquid had been shown to contain esterase activity which partially erodes the surface of the cuticle of barley leaves. The present investigation demonstrates that the liquid exudate contains cutinase activity. The presence of cutinase in the conidial exudate was confirmed by enzyme assays using 3H-cutin as the substrate. Like other fungal cutinases, the enzyme from E. graminis was inhibited by the serine esterase inhibitor diisopropyl fluorophosphate. The results are significant since they demonstrate for the first time the production of cutinase by a powdery mildew parasite. It is possible that the cutinase is involved in the erosion of the leaf cuticle which begins prior to the development of appressoria. Such erosion may be necessary either for recognition of the host surface by the appressorium or for adhesion and more efficient penetration of the leaf.

A natural killer cell granule protein that induces DNA fragmentation and apoptosis.

We report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called "fragmentin," was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe chromatin condensation within 1 h. Amino acid sequence of tryptic peptides indicated that fragmentin was highly homologous to the NK and T cell granule serine proteases RNK protease 1 and mouse cytotoxic T cell protease I (CCPI)/granzyme B. Preincubation with the serine esterase inhibitor 3,4-dichloroisocoumarin blocked fragmentin-induced DNA damage, but had no effect on cytolysin. Fragmentin activity against four lymphoma target cells was completely dependent on the presence of cytolysin. Fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses, suggesting that fragmentin activity was not limited to its effects on the nucleus. Fragmentin and cytolysin activity were completely inhibited by EGTA, indicating the process was Ca2+ dependent. A role for cytolysin in endocytosis of fragmentin was suggested by the observation that treatment of YAC-1 with cytochalasin B or sodium azide and 2-deoxyglucose blocked DNA fragmentation but not cytolysin activity. A 30-kD N alpha-CBZ-L-lysine thiobenzyl esterase, which copurified with fragmentin, was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin. Fragmentin activity was not dependent on protein synthesis, as cycloheximide treatment of YAC-1 cells did not prevent DNA damage. We postulate that fragmentin is the molecular mediator of NK cell-mediated DNA fragmentation and apoptosis.

Phenylmethanesulphonyl fluoride inhibits GPI anchor biosynthesis in the African trypanosome.

A wide variety of eukaryotic membrane proteins are anchored to the outside of cells by covalent linkage to glycosyl phosphatidylinositol (GPI). One of the best characterized examples is the variant surface glycoprotein (VSG) of the protozoan parasite, Trypanosoma brucei. The structure of the GPI precursor is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2-PI; the phosphoethanolamine moiety forms an amide linkage to the VSG polypeptide alpha-COOH group during its attachment to protein. Here we report that the serine esterase inhibitor, phenylmethanesulphonyl fluoride (PMSF), inhibits phosphoethanolamine incorporation into the GPI precursor resulting in the accumulation of a Man3GlcNH2-PI intermediate. PMSF exerts this effect both in living trypanosomes and in a trypanosome-derived cell-free system. This is the first report of an inhibitor which affects GPI biosynthesis but not N-glycosylation. A model of the mechanism of phosphoethanolamine incorporation into the GPI precursor, based on the known properties of PMSF, is presented.

Mechanisms of disruption of the articular cartilage surface in inflammation. Neutrophil elastase increases availability of collagen type II epitopes for binding with antibody on the surface of articular cartilage.

We recently observed that specific antibodies to type II collagen do not bind in appreciable amounts to the intact surface of articular cartilage, whereas antibodies to the minor collagen types V, VI, and IX do. These results suggest that the outermost cartilage surface layer prevented interaction of the antibodies with the major collagen type in articular cartilage. The present studies were designed to investigate the pathogenic mechanisms involved in the disruption of the cartilage surface layer in inflammatory arthritis. Articular cartilage obtained from rabbits undergoing acute antigen-induced arthritis of 72 h duration showed a significant increase in binding of anti-type II antibody to cartilage surfaces compared with normal control cartilage (P less than 0.01). Augmentation of anti-type II binding was also observed upon in vitro incubation of bovine articular slices or intact rabbit patellar cartilage for 1 h with human polymorphonuclear neutrophils (PMN), PMN lysates, or purified human PMN elastase. This increase was not inhibited by sodium azide, nor was it enhanced by incubation of cartilage with the strong oxidant hypochlorous acid. Chondrocyte-mediated matrix proteoglycan degradation in cartilage explants cultured in the presence of cytokines failed to increase antibody binding appreciably. The augmentation in antibody binding seen with PMN lysates was inhibited by the nonspecific serine-esterase inhibitor PMSF, but not by the divalent metal chelator EDTA. The elastase-specific inhibitor AAPVCMK also inhibited most of the PMN-induced increase in antibody binding, whereas the cathepsin G-specific inhibitor GLPCMK was much less effective. Incubation of intact cartilage with purified human PMN elastase indicated that this serine esterase could account for the increase in anti-type II collagen antibody binding to intact cartilage surfaces. These studies suggest that in an inflammatory response, PMN-derived elastase degrades the outer layer of articular cartilage, exposing epitopes on type II collagen. They also help clarify the pathogenic mechanisms involved in early articular cartilage damage in inflammatory joint diseases.

Involvement of a serine esterase in oxidant‐mediated activation of phospholipase A2 in pulmonary endothelium

Exposure of bovine pulmonary arterial endothelial cells to 1 mM H2O2 stimulated associated TAME-esterase and PLA2 activities. Pretreatment with the serine esterase inhibitors: PMSF (1 mM), DFP (1 mM), and alpha 1-PI (1 mg/ml) inhibited H2O2-induced stimulation of TAME-esterase and PLA2 activities. The TAME-esterase and PLA2 activities under H2O2 exposure were determined to be linearly correlated. Affinity labelling of the endothelial cell membrane with [3H]DFP demonstrated that the serine esterase resides in a protein having molecular weight of 29,000 daltons (29 kDa) which is similar to that of elastase. Treatment of the endothelial cell homogenate with trypsin (1 microgram/ml) also stimulated PLA2 activity.