Background and purpose:Toll-like receptor 4 (TLR4) expressed on spinal microglia and astrocytes has been suggested to play an important role in the regulation of pain signalling. The purpose of the present work was to examine the links between TLR4, glial activation and spinal release of prostaglandin E-2 (PGE(2)) and tumour necrosis factor (TNF), and the role these factors play in TLR4-induced tactile allodynia.Experimental approach:Toll-like receptor 4 was activated by intrathecal (i.t.) injection of lipopolysaccharide (LPS) and KDO2-Lipid A (KDO2) to rats. Tactile allodynia was assessed using von Frey filaments and cerebrospinal fluid collected through spinal dialysis and lumbar puncture. PGE(2) and TNF levels were measured by mass spectometry and elisa. Minocycline and pentoxifylline (glia inhibitors), etanercept (TNF-blocker) and ketorolac (COX-inhibitor) were given i.t. prior to injection of the TLR4-agonists, in order to determine if these agents alter TLR4-mediated nociception and the spinal release of PGE(2) and TNF.Key results:Spinal administration of LPS and KDO2 produced a dose-dependent tactile allodynia, which was attenuated by pentoxifylline, minocycline and etanercept but not ketorolac. Both TLR4 agonists induced the spinal release of PGE(2) and TNF. Intrathecal pentoxifylline blunted PGE(2) and TNF release, while i.t. minocycline only prevented the spinal release of TNF. The release of PGE(2) induced by LPS and KDO2 was attenuated by i.t. administration of ketorolac.Conclusions and implications:Activation of TLR4 induces tactile allodynia, which is probably mediated by TNF released by activated spinal glia.

Inhibitors of the Group IVA phospholipase A(2) (GIVA cPLA(2)) and GVIA iPLA(2) are useful tools for defining the roles of these enzymes in cellular signaling and inflammation. We have developed inhibitors of GVIA iPLA(2) building upon the 2-oxoamide backbone that are uncharged, containing ester groups. Although the most potent inhibitors of GVIA iPLA(2) also inhibited GIVA cPLA(2), there were three 2-oxoamide compounds that selectively and weakly inhibited GVIA iPLA(2). We further show that several potent 2-oxoamide inhibitors of GIVA cPLA(2) containing free carboxylic groups (Kokotos et al. J. Med. Chem. 2002, 45, 2891-2893) do not inhibit GVIA iPLA(2) and are, therefore, selective GIVA cPLA(2) inhibitors.

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.

The aim of this study was to examine, in biochemical detail, the functional role of the Arg152 residue in the selenoprotein Glutathione Peroxidase 4 (GPX4), whose mutation to His is involved in Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD). Wild-type and mutated recombinant enzymes with selenopcysteine (Sec) at the active site, were purified and structurally characterized to investigate the impact of the R152H mutation on enzymatic function. The mutation did not affect the peroxidase reaction's catalytic mechanism, and the kinetic parameters were qualitatively similar between the wild-type enzyme and the mutant when mixed micelles and monolamellar liposomes containing phosphatidylcholine and its hydroperoxide derivatives were used as substrate. However, in monolamellar liposomes also containing cardiolipin, which binds to a cationic area near the active site of GPX4, including residue R152, the wild-type enzyme showed a non-canonical dependency of the reaction rate on the concentration of both enzyme and membrane cardiolipin. To explain this oddity, a minimal model was developed encompassing the kinetics of both the enzyme interaction with the membrane and the catalytic peroxidase reaction. Computational fitting of experimental activity recordings showed that the wild-type enzyme was surface-sensing and prone to “positive feedback” in the presence of cardiolipin, indicating a positive cooperativity. This feature was minimal, if any, in the mutant. These findings suggest that GPX4 physiology in cardiolipin containing mitochondria is unique, and emerges as a likely target of the pathological dysfunction in SSMD.

Site-directed mutants of the group IA phospholipase A(2) from cobra venom were constructed and expressed in the methylotrophic yeast Pichia pastoris to probe for the proposed phosphatidylcholine (PC) activator site. Previous crystallographic and molecular modeling studies have identified two regions of the enzyme as likely candidates for this site. Residues Glu-55, Trp-61, Tyr-63, Phe-64, and Lys-65 were mutated to test the site advanced by Ortiz et al. [(1992) Biochemistry 31, 2887-2896] while Asp-23 and Arg-30 were mutated to assess the site proposed by Segelke et al. [(1998) J. Mol. Biol. 279, 223-232]. Expressed enzymes were purified by affinity chromatography and analyzed by SDS-PAGE, Western blotting, electrospray ionization mass spectroscopy, and circular dichroism. Both phospholipid headgroup specificity and rates of hydrolysis on monomeric PC substrates were determined and found to be similar for native, wild-type, and all of the mutant enzymes. These results suggest that all of the expressed enzymes were properly folded and contained functional catalytic sites. Mutations of the aromatic residues in the Ortiz site generally had little effect on PC activation, arguing against the importance of this region of the enzyme for PC activation; however, these aromatic amino acids appeared to be important for interfacial activation. In contrast, the D23N mutant in the Segelke site reduced PC activation by 10-fold without affecting activity toward micellar phosphatidylethanolamine substrates. Similar results were found with the D23N/R30M double mutant, suggesting that this region is critical for PC activation. These results provide evidence for the Segelke site as a PC activator site that is distinct from the catalytic site.

A lysophospholipase (LysoPLA I) has been purified and characterized from the mouse macrophage-like P388D(1) cell line (Zhang, Y. Y, and Dennis, E. A. (1988) J. Biol. Chem. 263, 9965-9972). This enzyme has now been sequenced, cloned, and expressed in Escherichia coli cells. The enzyme contains 230 amino acid residues with a calculated molecular mass of 24.7 kDa. It has a high helical content in its predicated secondary structure, which is also indicated in its CD spectrum. The cloned LysoPLA I was purified to homogeneity from the transformed E. coli cells by a gel filtration column and an ion exchange column, The specific activity of the purified protein is 1.47 mu mol/min mg toward 1-palmitoyl-sn-glycero-3-phosphorylcholine at pH 8.0 and 40 degrees C, corresponding to the reported value of 1.3-1.7 mu mol/min mg for the protein purified from the P388D(1) cells. In addition, the cloned protein cross-reacted with an antibody raised against LysoPLA I also purified from the P388D(1) cells. The deduced LysoPLA I sequence contains a well conserved GXSXG motif found in the active site of many serine enzymes, and the activity of the LysoPLA I was irreversibly inhibited by the classical serine protease inhibitor diisopropyl fluorophosphate. Furthermore, site-directed mutagenesis was employed to change Ser-119 in the GXSXG motif to an Ala. The resulting mutant protein lost all of its lysophospholipase activity, even though it had the same overall protein conformation as that of the wild-type LysoPLA I. Therefore, LysoPLA I has been demonstrated to be a serine enzyme with Ser-119 at the active site.

In recent years, considerable effort has been devoted to defining lipid signaling pathways, purification of the involved enzymes, and cloning of the genes encoding for these enzymes. However, much less is known about the mechanism, specificity of reaction, and the regulation of the catalytic activity of lipid-dependent enzymes and enzymes involved in generating lipid second messengers. The enzymes in lipid signaling pathways include lipid biosynthetic enzymes, lipid degrading enzymes, and enzymes that utilize lipid activators (cofactors).

The kinetics of the lysophospholipase purified from the P388D1 macrophage-like cell line (Zhang and Dennis (1988) J. Biol Chem. 263, 9965-9972) have been explored. Three different lysophospholipids were used in these studies: 1-hexadecanoyllysophosphatidylcholine, 1-tetradecanoyllysophosphatidylcholine, and 1-hexadecanoyllysophosphatidylglycerol. Since all of the substrate dependence data for these substrates fit a Hill model, the enzyme's activity appears to be cooperative requiring at least two lipid molecules for full enzymatic activity. The enzyme did not show a preference for any of these substrates since their kcat ranged from 1.2 to 1.5 mumol min-1 mg-1 and their half-maximal activities ([S]0.5) were achieved at substrate concentrations between 15 and 33 microM. Enzymatic activity also appeared to be independent of the aggregation state of the substrate. No dramatic changes in rate could be associated with substrate aggregation at the critical micelle concentration. This is in marked contrast to some phospholipases A2 that exhibit dramatic activations when their substrates aggregate. In very dilute solutions, less than 0.5 micrograms/ml protein, the lysophospholipase loses activity irreversibly within minutes. This effect of low protein concentrations can be overcome by maintaining the enzyme in the presence of greater than 10 microM lysophospholipid. This inactivation can affect kinetic studies, since the [S]0.5s for this enzyme are usually in this range. We have found that increasing the protein concentration with a 'non-specific protein', e.g., cytochrome c, can protect the enzyme without affecting activity and, thus, allow valid kinetic data to be obtained over the full substrate concentration range.

Various lipids have been implicated in signal transduction and as precursors of lipid mediators, among them arachidonic acid (Dennis et al., 1991). Production of the prostaglandins, thromboxanes, and prostacyclins via the cyclooxygenase pathway, as well as the production of the leukotrienes and lipoxins via the lipoxygenase pathway, all depend on the availability of free arachidonic acids. As illustrated in Figure 1, control of the production of free arachidonic acid is believed to involve an as yet undefined membrane receptor event (Dennis, 1989). Presumably this event involves the activation of a phospholipase, either directly or through mediators. Presumably this phospholipase is membrane associated or becomes so after translocation (Channon and Leslie, 1990). Because the bulk of cellular arachidonic acid is found esterified in the sn-2position of membrane phospholipids, a phospholipase A2 is the simplest and most logical enzyme

Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is approximately as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous. The spectrophotometric SIBLINKS assay and some of the fluorescent assays show promise of filling this need.

This chapter focuses on the technique of radiation inactivation, which can determine the size and aggregation state of a macromolecule even in a sea of phospholipid in intact cells. It also summarizes the theoretical aspects of this procedure, including its advantages and some precautions that must be taken to ensure the proper interpretation of the results. Protein molecular size determination using radiation inactivation is performed by exposing protein samples to increasing doses of ionizing radiation. The principal experimental requirement is that the irradiation must be conducted on frozen samples and thus the activity of interest must survive freezing and thawing. After irradiation, samples are assayed. Any functional or structural property of the protein can be measured including enzymatic activity, ligand binding to receptors or even the remaining protein structure or fluorescence. The loss of activity or structure with increasing radiation dose is analyzed according to target theory to give a target size for the protein. This technique has also been applied to complex systems involving regulatory units and biological functions as complex as muscle contraction.

Phospholipids comprise a large portion of cell membranes and serve to form the hydrophobic barrier between the internal and external environments. Their regulation is crucial for membrane integrity and cell viability. Phospholipid catabolism plays an important role in this regulatory process. It has recently been found to have another, equally important, cellular function. Many of the breakdown products of phospholipid catabolism are important components in a number of regulatory pathways. These include the inositol phosphate second messengers, which are degradation products of phosphatidylinositol, and the eicosanoids, which are products of arachidonic acid metabolism (1). Phospholipid catabolism is carried out by four basic types of enzymes that hydrolyze the four ester bonds found in phospholipids. These enzymes are the phospholipases A1, A2, C, and D (2). Phospholipases A1 and A2 are responsible for cleaving the fatty acid ester bonds while phospholipases C and D cleave at the headgroup phosphoester bonds.

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)

Experiments were carried out to determine whether the hydrolytic step or the product release step is the rate-limiting step for non-activated phospholipase A2 hydrolysis (Dennis, E.A. (1983) in The Enzymes, 3rd Edn., Vol 16 (Boyer, P., ed.), pp. 307-353, Academic Press, New York) of mixed micelles of phosphatidylethanolamine and Triton X-100 in the absence of activator phospholipids and of monomeric short chain phosphatidylcholine in the absence of an interface (Lombardo, D. et al. (1986) J. Biol. Chem. 261, 11663-11666). Phospholipase A2-catalyzed exchange of H2(18)O into 1-alkyl-2-[1(13)C]lauroyl-sn-glycero-3-phosphorylethanolamine and into 1-hexanoyl-2-[1-13C]hexanoyl-sn-glycero-3-phosphorylcholine were examined. Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C-NMR. Both the substrate and products of the reactions were examined for 18O incorporation. 18O was incorporated into the fatty acid product, but no incorporation of 18O into the substrate was found. These results suggest that the hydrolytic step is not followed by a higher energy transition state and that it, or a step before it, is rate-limiting. Coupled with kinetic experiments, this strongly suggests that the hydrolytic step is the rate-limiting step. Thus, the role of micellar and membrane interfaces in phospholipase A2 reactions does not appear to be by aiding product removal from the enzyme active site.

The kinetics of the Ca2+-dependent, alkaline pH optimum, membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line were studied using various phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates. This enzyme exhibits "surface dilution kinetics" toward PC in Triton X-100 mixed micelles, and the "dual phospholipid model" was found to adequately describe its kinetic behavior. With substrate in the form of sonicated vesicles, the dual phospholipid model should give rise to Michaelis-Menten type kinetics. However, the hydrolysis of dipalmitoyl-PC, 1-palmitoyl-2-oleoyl-PC, and 1-stearoyl-2-arachidonoyl-PC vesicles exhibited two distinct activities. Below 10 microM, the data appeared to follow Michaelis-Menten behavior, while at higher concentrations, the data could best be fit to a Hill equation with a Hill coefficient of 2. These PCs had Vmax values for the low substrate concentration range of 0.2-0.6 nmol min-1 mg-1 and Km values of 1-2 microM. At the high substrate concentration range, the Vmax values were between 5 and 7 nmol min-1 mg-1. PC containing unsaturated fatty acids had an apparent Km, determined from the Hill equation, of about 15 microM, while the apparent Km of dipalmitoyl-PC was 0.6 microM. When 70% glycerol was included in the assays, a single Michaelis-Menten curve was obtained for both dipalmitoyl-PC and 1-stearoyl,2-arachidonoyl-PC. Possible explanations for these kinetic results include reconstitution of the membrane-bound phospholipase A2 in the phospholipid vesicle or the enzyme has tow distinct phospholipid binding function. The kinetics for both dipalmitoyl-PC and dipalmitoyl-PE hydrolysis in vesicles was very similar, indicating that the enzyme does not greatly prefer one of these head groups over the other. The enzyme also showed no preference for arachidonoyl containing phospholipid. Enzymatic activity toward PC containing saturated fatty acids was linear to about 15% hydrolysis while the hydrolysis of PC containing unsaturated fatty acids was linear to only about 5%. This loss of linearity was due to inhibition by released unsaturated fatty acids. Arachidonic acid was found to be a competitive inhibitor of dipalmitoyl PC hydrolysis with a K1 of 5 microM. This tight binding suggests a possible in vivo regulatory role for arachidonic acid. Three compounds of the arachidonic acid cascade, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, showed no inhibition of enzymatic activity.

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.

Manoalide, a natural product from sponge, displays anti-inflammatory activity in vivo. Previous work has shown that manoalide is also a potent covalent inhibitor of the extracellular phospholipase A2 from cobra venom and that the inhibition correlated with a pH-dependent change in manoalide (Lombardo and Dennis (1985) J. Biol. Chem. 260, 7234–7240). Manoalide contains two rings and the opening of either would produce an α,β-unsaturated aldehyde. The cobra venom phospholipase A2 may be able to catalyze the opening or isomerization of one of these rings, raising the possibility that manoalide is acting as a suicide substrate. To ascertain the role of the γ-lactone ring in the inhibition, we have now investigated a synthetic manoalide analogue, 3(cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide (HDHB) which contains only the α,β-unsaturated γ-lactone ring. We have found that the closed and open forms are in rapid equilibrium between pH 4 and 9 with the cyclic form being preferred at acidic pH values and the open cis form preferred at pH 9.5. When the pH is raised above 12, the α,β double bond isomerizes to form trans-HDHB. Once the trans compound is formed, it is stable at all pH values and does not recyclize to the γ-lactone ring. The observed pKa of 7.7 found for the inhibition of manoalide agrees well with the transition of the closed to the cis form of the γ-lactone ring. Kinetic experiments with the HDHB compound show that under conditions in which the cis and closed form of the inhibitor are present in equal molar ratios, HDHB is not an irreversible inhibitor, but reversibly competes with substrate. However, the kinetics of this inhibition are complex and do not follow either pure competitive or non-competitive inhibition. The trans-HDHB exhibits similar complex kinetics but is several times more potent. The distinct differences between the behavior of manoalide and HDHB clearly indicate that while the γ-lactone ring may play an important role in manoalide inhibition, it alone does not produce irreversible inhibition.