Low- and high-Km transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytes.

Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside-out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio-3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high-affinity transporter for DNP-SG and GSSG in erythrocytes is suggested.     

Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. Effects of manoalide, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacyl bromide

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.     

A continuous spectrophotometric assay for phospholipase A(2) activity

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).     

Characterization of extracellular phospholipase A2 in rheumatoid synovial fluid

Phospholipase A2 (PLA2) activity has now been identified in rheumatoid synovial fluids. This PLA2 is a calcium-requiring protein of MW 11,000 with a neutral pH optimum. Its activity was inhibited by high concentrations of Mg2+, and by the active site-directed histidine reagent p-bromophenacyl bromide. Ionic and nonionic detergents, or the sulfhydryl reagent dithiothreitol caused loss of enzyme activity. Synovial fluid PLA2 did not interact with sulphated mucopolysaccharides such as heparin or chondroitin sulphate. Release and sequestration of PLA2 in the joint space may contribute to the characteristic rheumatoid inflammatory changes.     

Phospholipid binding and the activation of group IA secreted phospholipase A2

Equilibrium dialysis was used to study the binding of two nonhydrolyzable, short chain phospholipid analogues to the secreted group IA phospholipase A(2) (PLA(2)), which has been shown to contain several phospholipid binding sites that dramatically affect activity. This study provides new insight into how these activations occur. One analogue contained a phosphorylethanolamine (DiC(6)SNPE) headgroup, while the other contained a phosphorylcholine (DiC(6)SNPC) headgroup. Using phospholipase D, we incorporated tritium into each analogue. No binding of DiC(6)SNPE to PLA(2) was observed under submicellar conditions. Addition of submicellar amounts of Triton X-100 resulted in a linear nonsaturating response to lipid concentration, suggestive of premicellar aggregation of the DiC(6)SNPE with Triton X-100 and PLA(2). Binding of DiC(6)SNPE when presented as Triton X-100 mixed micelles saturated at 0.93 binding sites per PLA(2) with a K(D) of 38 microM. Addition of sphingomyelin, a potent activator of PLA(2) hydrolysis of phosphorylethanolamine containing compounds, resulted in a 13-fold decrease in the K(D), to 2.8 microM. This suggests that changes in the catalytic site binding affinity contribute to "phosphatidylcholine activation". Binding of DiC(6)SNPC with 2.0 mM Triton X-100 showed positive cooperativity (Hill coefficient of 1.7), which saturated at 2.0 binding sites per PLA(2). No binding of either analogue was observed when the catalytic site was alkylated with p-bromophenacyl bromide. Since p-bromophenacyl bromide does not physically block the phosphatidylcholine activator site, this indicates that the two phosphatidylcholine binding sites interact. The binding studies show that DiC(6)SNPC binds cooperatively to two sites on group IA PLA(2), while DiC(6)SNPE binds to only one site.     

Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study

We examined whether phospholipase A₂ (PLA₂) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius , using inhibitors and an activator of this enzyme. Quinacrine and p-bromophenacyl bromide (PBPB) inhibited the egg jelly-induced acrosome reaction at 100 μM, but not the ionomycin-induced one. Depression of egg jelly-induced increase of intracellular free Ca²⁺concentration ([Ca²⁺]_i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA², caused a [Ca²⁺]_i increase and a formation of acrosomal process-like structure on sperm head. These results suggest that PLA₂ participates in the early step(s) of the acrosome reaction of sea urchin sperm.     

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.     

Modulation of macrophage interaction with Trypanosoma cruzi by phospholipase A2-sensitive components of the parasite membrane

The presence of phospholipase A2 (PLA2) significantly increased the association between Trypanosoma cruzi and macrophages. This effect reflected alterations to the parasite membrane since it was reproduced only when the parasite but not the macrophage was pretreated with PLA2. That PLA2 activity was responsible for the noted enhancement was indicated by the ability of the specific substrate phosphatidylcholine to block it. The presence of the PLA2 inhibitors quinacrine, 4-bromophenacyl bromide or phentermine markedly inhibited parasite-macrophage association. Quinacrine also inhibited association of the parasite with a non-phagocytic host cell. These results suggested a role for endogenous PLA2 in the initial stages of cell infection by T. cruzi.     

Purification and characterization of a cytosolic phospholipase A2 from rat liver

A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.     

Role of human sperm phospholipase A2 in fertilization: effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration.

Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined. Highly purified enzyme hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli optimally at neutral to alkaline pH with 5 mM CaCl2 and 150 mM NaCl (specific activity = 20 mumol/min/mg). Activity was inhibited in a dose-dependent manner by an oligomer of prostaglandin B1 (IC50 = 1.5 microM) reported to inhibit human phospholipases A2 in vitro and in situ. Sperm phospholipase A2 injected into mouse foot pad induced a dose-dependent edema that was inhibited by oral administration of prostaglandin Bx (IC50 < or = 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide. Human sperm phospholipase A2 (10 micrograms) induced fusion of phosphatidylserine vesicles in the presence of 1 mM calcium chloride by approximately 80% (+/- 10%) as determined by monitoring turbidity (O.D.400) and efficiency of fluorescence resonance energy transfer. This enzyme-induced fusion was accompanied by phospholipid hydrolysis, and both fusion and phospholipid degradation were inhibited by more than 60% when enzyme was preincubated with 5 microM prostaglandin Bx. Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin Bx (IC50 approximately 15 microM) during capacitation; sperm motility was not affected by this treatment. Capacitation in the presence of prostaglandin Bx had little to no effect on the in vitro acrosome reaction. These results suggest that sperm phospholipase A2 and its modulators may contribute to membrane fusion events in mammalian fertilization.     

Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.     

Studies on phospholipase A2 in human seminal plasma.

1. Human seminal plasma and posterior lobe of prostate was found to have phospholipase A2 (PLA2) activity hydrolysing phosphatidylethanolamine with 14C-labelled linoleic and arachidonic acid. 2. A negative relationship was between sperm count and PLA2 activity in human seminal plasma. 3. The purified PLA2 from human seminal plasma showed high affinity to heparin, sensitivity toward p-bromophenacyl bromide, Pb2+, dithioerythritol and EDTA and it was activated by Ca2+ and Mn2+. 4. The purified PLA2 had alkaline pH optimum (7.5-10.0) and pI-value of 5.3. In SDS-PAGE enzyme preparation resulted in two bands with mol. wt of 14,000 and 16,000.     

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms

The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.     

Phospholipase A of Guinea Pig Spermatozoa: Its Preliminary Characterization and Possible Involvement in the Acrosome Reaction

Phospholipase A activity was demonstrated in guinea pig spermatozoa using [U-¹⁴C] phosphatidyl choline as a substrate. The activity had a neutral pH optimum, was stimulated by Ca²⁺ and low concentrations of detergent, and. was inhibited by EDTA, mepacrine and p-bromophenacyl bromide. Appropriate concentrations of mepacrine and p-bromophenacyl bromide inhibited the acrosome reactions of capacitated spermatozoa without interfering with their motility. These results support the notion that phospholipase A is involved in the acrosome reaction of mammalian spermatozoa.     

Involvement of phospholipase A2 activation in anthrax lethal toxin-induced cytotoxicity

The molecular mechanism of cytotoxic effect exerted by the lethal toxin (LeTx) of Bacillus anthracis is not well understood. In the present study, using primary culture of mouse peritoneal macrophages, we have investigated possible cytotoxic mechanisms. LeTx was not found to induce high levels of nitric oxide (NO) production for NO-mediated toxicity. Fragmentation of DNA, a biochemical marker of apoptosis, was not observed in LeTx-treated cells. Pretreatment of cells with antioxidants such as melatonin and dehydroepiandrosterone (DHEA) did not protect the LeTx-induced cytotoxicity. However, addition of phospholipase A2 (PLA2) inhibitors (quinacrine, p-bromophenacyl bromide, manoalide, butacaine) to the culture medium resulted in the inhibition of cytotoxicity of LeTx in a dose-dependent manner. LeTx-induced cytotoxicity was also inhibited by the tyrosine-specific protein kinase inhibitor genistein, but not by the protein kinase C inhibitors staurosporine or H-7. The results of these studies indicate a role for PLA2 and protein kinase in the cytotoxic mechanism of macrophages by anthrax lethal toxin.     

Translocation of phospholipase A2 from cytosol to membranes in rat brain induced by calcium ions

Phospholipase A2 (PLA2) activities were found in the cytosolic fractions of rat brain. Using the gel filtration chromatography, two major peaks of PLA2 activities were demonstrated: PLA2-H (200-500 kDa) and PLA2-L (100 kDa). PLA2-L was active at both neutral and alkaline pH and absolutely required Ca2+ for the activity, while the activity of PLA2-H was detected only at alkaline pH and independent of Ca2+. The activation of PLA2-L by Ca2+ was biphasic; the first observed at 1-100 microM Ca2+ and the second at 10 mM Ca2+. In the reconstitution system of partially purified PLA2-L and synaptosomal membranes from rat brain, PLA2-L associated with the membranes in a Ca2(+)-dependent manner. The association was completed within 5-10 min at 25 degrees C both at 10 microM and 1 mM Ca2+, though amount of PLA2-L translocated was dependent on Ca2+ concentrations. These results suggest that Ca2+ promotes the translocation of the cytosolic PLA2-L to membranes where phospholipids, substrate of PLA2, are present.     

Regulation of lung surfactant secretion by phospholipase A2

Arachidonic acid has been shown to stimulate lung surfactant secretion from alveolar epithelial type II cells. To identify the (phospho)lipases responsible for generating arachidonic acid during lung surfactant secretion, the effects of various (phospho)lipase inhibitors on phosphatidylcholine (PC) secretion from rat alveolar type II cells were investigated. N-(p-amylcinnamoyl)anthranilic acid (ACA), a general inhibitor of phsopholipase A2 (PLA2), inhibited ATP-stimulated PC secretion in a dose-dependent manner. ACA also blocked PC secretion from type II cells stimulated by other secretagogues including phorbol 12-myristate 13-acetate, Ca2+ ionophore A23187 and terbutaline, indicating that PLA2 acts at a late step distal to the generation of second messengers. To determine which PLA2 isoform(s) is involved in lung surfactant secretion, selective inhibitors to different types of PLA2 were used to inhibit PLA2 activity in type II cells. The cytosolic PLA2 (cPLA2) inhibitor, arachidonyl trifluoromethyl ketone, was found to inhibit ATP-stimulated PC secretion, whereas the secretory PLA2 inhibitors, oleoyloxyethylphosphocholine, aristolochic acid, or p-bromophenacyl bromide, and the Ca2+-independent PLA2 inhibitors, palmitoyl trifluoromethyl ketone, or haloenol lactone suicide substrate, had no effect. In addition to PLA2, arachidonic acid is released from diacylglycerol (DAG) by DAG and monoacylglycerol lipases. The DAG lipase inhibitor, RHC-80267 also blocked ATP-stimulated PC secretion. The results suggest that both pathways for generating arachidonic acid via cPLA2 and DAG lipase may participate in lung surfactant secretion.     

Inhibition of phospholipase A2 by heparin

Phospholipase A2 (PLA2) is an important enzyme in the regulation of cell behavior. The hydrolysis of phosphatidylcholine in vitro catalyzed by porcine pancreatic PLA2 was inhibited by heparin. Other glycosaminoglycans inhibited PLA2 activity to a significantly lesser extent, with a pattern of inhibition: heparin much greater than chondroitin sulfate (CS)-C greater than CS-A greater than CS-B greater than keratan sulfate. Hyaluronic acid and heparan sulfate caused no inhibition. Heparin's ability to inhibit PLA2 activity did not depend on substrate concentration, but did depend on ionic strength, with inhibition decreasing with increasing ionic strength. Heparin inhibition also varied with pH, being more effective at pH 5-8 than at pH 10. As a consequence, heparin induced a shift of the pH optimum of PLA2 from 7 to 8. Histone IIA and protamine sulfate, heparin-binding proteins, reversed heparin-induced PLA2 inhibition. The concentration of heparin which inhibited PLA2 activity by 50% increased with increasing enzyme concentration. Furthermore, PLA2 bound to heparin-Affigel. The data indicate that the catalytic potential of PLA2 can be regulated by heparin or heparin-like molecules and that inhibition is contingent on the formation of a heparin-PLA2 complex.     

磷脂酶A2在内毒素致大鼠肺损伤中的作用

大鼠静脉注射大肠杆茵内毒素（３０ｍｇ／ｋｇ）后３ｈ肺血管外水量和支气管肺泡灌洗液中蛋白浓度明显增加,表明发生了通透性肺水肿;同时血清和支气管肺泡灌洗液中磷脂酶Ａ２（ＰＬＡ２）活性升高,且ＰＬＡ,活性的升高与肺血管外水量的增加呈显著正相关。预先给予ＰＬＡ２抑制剂对溴苯酰基溴可抑制内毒素引起的ＰＬＡ２活性升高和通透性肺水肿。提示ＰＬＡ２介导了内毒素引起的肺损伤。