Modulation of eicosanoid biosynthesis and inhibition of substrate availability for phospholipase A2 by a modified hexose sugar, amiprilose hydrochloride

The modified hexose, sugar, amiprilose HCl [1, 2-O-isopropylinine-3-O-3′-(N′,N′-dimethylamino-n-propyl)-D-glucufuranose hydrochloride], has previously been shown to have antiinflamatory acitivies. The present study assessed whether eicosanoid biosynthesis is regulated by amiprilose HCl adn whether the regulation is influenced at the early stage of arachidonate liberation from the phospholipid by phospholipase A₂ (PLA₂). Secretiion of both prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) by peritoneal macrophages and neutrophils from amiprilose HCl-treated mice was reduced with neutrophils being slightly more sensitive to the inhibitory effects. Amiprilose HCl was less effective at inhibiting PGE₂ and LTB₄ secretion that it was . Amiprilose HCl did not have a direct inhibitory effect on the PLA₂ enzyme or on secretion of the soluble form od PLA₂. In contrast, amiprilose HCl modulated the phospholipid substrate for PLA₂ as there was inhibition of label release from [1-¹⁴C]-oleic acid-labeled substrate source (i) when labeled substrate for pure PLA₂ had been preincubated with amiprilose HCl, or (ii) when labeled peritoneal cells, which had been preincubated with amiprilose HCl, were used as a substrate source either for pure PLA₂ or for their own PLA₂. Amiprilose HCl was found to bind to peritoneal cells rapidly, but transiently, with maximal binding occurring within 5 min at 37°C. Thus, amiprilose HCl was shown to be inhibitory to secretion of PGE₂ and LTB₄, at least in part, by inhibiting the availability of substrate for PLA₂.     

Lysosomotropic agents activate the capacity for calcium dependent pinocytosis in starvedAmoeba proteus: evidence for a mechanism involving phospholipase A2

Summary Pinocytosis induced by Na⁺ was assayed by phase contrast microscopy in 8–12 days starvedAmoeba proteus. These cultures were inactive with respect to calcium-dependent Na⁺-induced pinocytosis, but treatment with amino acid methyl and ethyl esters increased their capacity for pinocytosis. Besides promoting pinocytosis these compounds also stimulated calcium-sensitive secretion of lysosomal enzymes from normal, 2–3 days starved, cells. Only uncharged 1-forms of the amino acid esters were effective. Also other lysosomotropic compounds including monodansylcadaverine, glycine-phenylalanine-2-naphthylamide, NH₄Cl, and the ionophores monensin and A23187 activated starved cells. The effect of these agents (except A23187) was inhibited by the drug dantrolene suggesting that activation is a consequence of release of Ca²⁺ from intracellular stores. Several of the lysosomotropic agents also lost their activating effect in the presence of phospholipase A₂ (PLA₂) inhibitors. To investigate whether or not PLA₂ activity in the cell culture could imitate the effect of the lysosomotropic agents, we incubated starved cells with snake venom PLA₂s. These enzymes caused rapid, dantrolene-sensitive activation of the cells. Measurement of endogenous PLA₂in normal cells revealed significant cellular activity but no significant secretion of the enzyme into the culture medium was observed. Together the studies with enzyme inhibitors and dantrolene suggest that the process by which lysosomotropic agents affect pinocytosis involves activation of PLA₂ and release of Ca²⁺ from intracellular stores.Abbreviations AnBOMe amino-n-butyric acid methylester - Et ethylester - GPN glycine-1-phenylalanine-2-naphthylamide - MDC monodansylcadaverine - MDTC monodansylthiacadaverine - Me methylester - pBPB p-bromo phenacylbromide - PLA₂ phospholipase A₂     

Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A₂ (PLA₂) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA₂ in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA₂ activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA₂ activity in ras-transformed cells compared to the corresponding palmitatestearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA₂ activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA₂ in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.     

Phospholipase A2 in hemocytes of the tobacco hornworm,Manduca sexta

On the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A₂ (PLA₂) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLA₂ hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PLA₂s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA₂ activity was sensitive to hemocyte homogenate protein concentration (up to 250 μg protein/reaction), pH (optimal activity at pH 8.0), and the presence of a Ca²⁺ chelator. Like PLA₂s from mammalian sources, the hemocyte PLA₂ was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA₂s require Ca²⁺ for catalytic activity, some PLA₂s, including the hemocyte enzyme, are Ca²⁺-independent. The hemocyte PLA₂ exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA₂ that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PLA₂ may be the first biochemical step in synthesis of the eicosanoids that mediate cellular immunity in insects. © 1996 Wiley-Liss, Inc.     

Ontogeny of gene expression of group IB phospholipase A₂ isoforms in the red sea bream, Pagrus (Chrysophrys) major

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A₂ (PLA₂) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA₂s, the present study investigated the localization and expression of DE-1 and DE-2 PLA₂ genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA₂ mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA₂ mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA₂ mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA₂ mRNA was highly expressed in pancreatic tissue, and DE-2 PLA₂ mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA₂ mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA₂ proteins.     

Phospholipase A2 and Its Role in Brain Tissue

Abstract: Phospholipase A₂ (PLA₂) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA₂-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA₂ in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA₂ has during both physiologic and pathologic states. Several different PLA₂ enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA²⁺-dependent and Ca²⁺-independent, based on their catalytic dependence on Ca²⁺. Under physiologic conditions, PLA₂ may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA₂ activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA₂ found in the central nervous system and attempts to define the role of PLA₂ during both physiologic and pathologic conditions.     

Effects of exogenous phospholipases on brain membrane phospholipid perturbation, (Na+ + K+)-ATPase activity and cellular swelling of brain slices

Rat brain membranes were incubated with bee venom phospholipase A₂ (PLA₂) or phospholipase C (PLC) from Clostridium perfringens. PLA₂ caused a significant increase in free polyunsaturated fatty acids concomitant with membrane phospholipid degradation as monitored by HPLC and by gas chromatography. Equal concentrations of PLC had a much lesser effect than PLA₂. Divergent and differential effects were shown on deacylation and incorporation of [³H]arachidonic acid in membrane phospholipids. The incorporation of [³H]arachidonic acid into various phospholipids was greatly reduced by PLA₂ (0.018 units/ml) whereas PLC at identical concentration was not effective. PLA₂ inhibited (Na⁺ + K⁺)-ATPase but was not effective on p-nitrophenyl-phosphatase activity whereas PLC stimulated both enzymes. PLA₂ induced swelling of cortical brain slices whereas PLC was not effective. Thus, the severity of the perturbation of membrane integrity, and the inhibition of (Na⁺ + K⁺)-ATPase in brain membranes may play an important role in cellular swelling of brain slices induced by PLA₂.     

Phospholipase A2 activity in the fat body of the tobacco hornworm Manduca sexta

We report on phospholipase A₂ (PLA₂) activity in homogenates prepared from fat bodies of the tobacco hornworm Manduca sexta. PLA₂ activity is responsible for hydrolyzing fatty acids from the sn-2 position of phospholipids. The rate of hydrolysis increased with increasing homogenate protein concentration up to ～? 320 μg protein/ml reaction volume. Higher protein concentrations did not appreciably increase the rate of PLA₂ activity. As seen in some, but not all PLA₂s from mammalian sources, hydrolyzing activity increased linearly with time. The fat body activity was sensitive to pH (optimal activity at pH 8–9) and temperature (optimal activity at ～?40°C). The activity was associated with fat body rather than hemolymph, because no activity was detected in cell-free serum. The fat body PLA₂ activity differs from the majority of PLA₂s with respect to calcium requirements. Whereas most PLA₂s are calcium-independent. A few others are known to require submicromolar calcium concentrations. The fat body activity appears to be calcium independent. These data show that a PLA₂ activity that can hydrolyze arachidonic acid from the sn-2 position of phospholipids is associated with the tobacco hornworm fat body. The biological significance of this activity relates to biosynthesis of eicosanoids. Pharmacological inhibition of PLA₂ impairs the ability of this insect to respond to bacterial infections. Since the impairment can be reversed by treatment with exogenous arachidonic acid, the PLA₂ activity may be an important step in eicosanoid biosynthesis. © 1993 Wiley-Liss, Inc.     

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.     

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A₂ (PLA₂). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA₂ toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA₂. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA₂. Moreover, it was found that PLA₂ and N-terminally mutated PLA₂ adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA₂ and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA₂, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA₂ at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA₂.     

Three metabolites from an entomopathogenic bacterium,Xenorhabdus nematophila,inhibit larval development of Spodoptera exigua (Lepidoptera: Noctuidae) by inhibiting a digestive enzyme,phospholipase A2

Abstract An entomopathogenic bacterium, Xenorhabdus nematophila, has been known to induce significant immunosuppression of target insects by inhibiting immune‐associated phospholipase A₂ (PLA₂), which subsequently shuts down biosynthesis of eicosanoids that are critical in immune mediation in insects. Some metabolites originated from the bacterial culture broth have been identified and include benzylideneacetone, proline‐tyrosine and acetylated phenylalanine‐glycine‐valine, which are known to inhibit enzyme activity of PLA₂ extracted from hemocyte and fat body. This study tested their effects on digestive PLA₂ of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrations of the three metabolites resulted in significant adverse effects on larval development even at doses below 100 μg/mL. In particular, they induced significant reduction in digestive efficiency of ingested food. All three metabolites significantly inhibited catalytic activity of digestive PLA₂ extracted from midgut lumen of the fifth instar larvae at a low micromolar range. These results suggest that the inhibitory activities of the three bacterial metabolites on digestive PLA₂ of S. exigua midgut may explain some of their oral toxic effects.     

Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B(2), PGD(2), PGE(2) and PGF(2alpha), all of which were confirmed by analysis on gas chromatography--mass spectrometry (GC--MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA(2) for this product. PGA(2) was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22 degrees C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology.     

Arachidonic acid release from NIH 3T3 cells by group-I phospholipase A2: Involvement of a receptor-mediated mechanism

Group I pancreatic phospholipase A₂ (PLA₂ I) is primarily a digestive enzyme. Recently, however, in addition to its catalytic activity a receptor-mediated function has been described for this enzyme. PLA₂ I binding to its receptor induces cellular chemokinesis, proliferation, and smooth muscle contraction. This enzyme also induces the production of prostaglandin E₂ in certain cells and may have a proinflammatory role. However, despite its ability to hydrolyze phospholipids in in vitro assays, PLA₂-I does not efficiently catalyze release of AA from intact cells. Here, we demonstrate that while short-term exposure of NIH 3T3 cells to PLA₂-I is ineffective, exposure of 6 h or longer significantly increases the basal release of AA. Dose-response curve of PLA₂-I-induced AA release was saturable with an EC₅₀ of 14.01 ± 1.36 nM (n = 3). [³H]-AA was preferentially released over [³H]-oleic acid by PLA₂-I, inactivated with 4-bromophenacyl bromide, was fully capable of mediating AA release. These data suggest that a non-catalytic, receptor-mediated mechanism is involved in PLA₂-I-induced AA release in NIH-3T3 cells. This relase of AA is not dependent on protein kinase C or Ca²⁺ concentration. Comparison of the effect of PLA₂-I with those of ATP and platelet-derived growth factor indicates that each of these agonists regulates AA release via independent pathways. Neither the basal enzymatic activity of the 85-kDa cytosolic PLA₂ nor the protein level of this enzyme was affected by treatment of cells with PLA₂-I. However, the increase in basal enzymatic activity of 85 kDa PLA₂ due to protein kinase C activation was further enhanced by pretreatment of cells with PLA₂-I. We conclude that: (1) short-term exposure of cells to PLA₂ I does not cause measurable AA release; (2) release of AA from intact cells by this enzyme requires long-term exposure; (3) AA release is not mediated by a direct catalytic effect of PLA₂ I; and (4) AA release by PLA₂ I is accomplished via a receptor-mediated process. Taken together, these results raise the possibility that PLA₂ I, in addition to its digestive function, may also contribute to aggravate preexisting inflammatory processes and/or to initiate new ones when chronic exposure of cells to this enzyme occurs. © 1995 Wiley-Liss Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Phospholipase A2 and its Molecular Mechanism after Spinal Cord Injury

Phospholipases A₂ (PLA₂s) are a diverse family of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor which have been implicated in pathological states of numerous acute and chronic neurological disorders. To date, more than 27 isoforms of PLA₂ have been found in the mammalian system which can be classified into four major categories: secretory PLA₂, cytosolic PLA₂, Ca²⁺-independent PLA₂, and platelet-activating factor acetylhydrolases. Multiple isoforms of PLA₂ are found in the mammalian spinal cord. Under physiological conditions, PLA₂s are involved in diverse cellular responses, including phospholipid digestion and metabolism, host defense, and signal transduction. However, under pathological situations, increased PLA₂ activity, excessive production of free fatty acids and their metabolites may lead to the loss of membrane integrity, inflammation, oxidative stress, and subsequent neuronal injury. There is emerging evidence that PLA₂ plays a key role in the secondary injury process after traumatic spinal cord injury. This review outlines the current knowledge of the PLA₂ in the spinal cord with an emphasis being placed on the possible roles of PLA₂ in mediating the secondary SCI.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24 h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.     

PHOSPHOLTPID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELLS FROM BRAINS OF 17-DAY OLD RATS1

Neuronal and glial cells were isolated from the brains of 17-day old rats and incubated for 5 h with either radioactive inorganic phosphate, palmitate, serine, choline or ethanolamine in a tissue culture medium. A comparison of the results suggests that both neuronal and glial cells exhibit effective de novo, phospholipid synthesis and that the observed differences in the uptake are due more to quantitative rather than qualitative differences in phospholipid metabolism of both cell types. Incubations of the combined neuronal and glial fractions with ³²PO₄ and [³H]palmitate result in incorporations up to 100% higher than calculated from incubations of the separate fractions, suggesting that phospholipid metabolism of neuronal and glial cells may exhibit cooperativity.     

Eicosanoids mediate insect hemocyte migration

Hemocyte migration toward infection and wound sites is an essential component of insect defense reactions, although the biochemical signal mechanisms responsible for mediating migration in insect cells are not well understood. Here we report on the outcomes of experiments designed to test the hypotheses that (1) insect hemocytes are able to detect and migrate toward a source of N-formyl-Met-Leu-Phe (fMLP), the major chemotactic peptide from Escherichia coli and (2) that pharmaceutical modulation of eicosanoid biosynthesis inhibits hemocyte migration. We used primary hemocyte cultures prepared from fifth-instar tobacco hornworms, Manduca sexta in Boyden chambers to assess hemocyte migration toward buffer (negative control) and toward buffer amended with fMLP (positive control). Approximately 42% of negative control hemocytes migrated toward buffer and about 64% of positive control hemocytes migrated toward fMLP. Hemocyte migration was inhibited (by >40%) by treating hornworms with pharmaceutical modulators of cycloxygenase (COX), lipoxygenase and phospholipase A2 (PLA2) before preparing primary hemocyte cultures. The influence of the COX inhibitor, indomethacin, and the glucocorticoid, dexamethasone, which leads to inhibition of PLA2, was expressed in a dose-dependent way. The influence of dexamethasone was reversed by injecting arachidonic acid (precursor to eicosanoid biosynthesis) into hornworms before preparing primary hemocyte cultures. The saturated fatty acid, palmitic acid, did not reverse the inhibitor effect. These findings support both our hypotheses, first that insect hemocytes can detect and respond to fMLP, and second, that insect hemocyte migration is mediated by eicosanoids.     

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.