Preparation of the Full Set of Recombinant Mouse- and Human-Secreted Phospholipases A2.

A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2 (sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2 was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles.

A pyrrolidine-based specific inhibitor of cytosolic phospholipase A(2)alpha blocks arachidonic acid release in a variety of mammalian cells.

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A(2): structure-function studies with vesicle, micelle, and membrane assays.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

The inhibition of protein prenyltransferases by oxygenated metabolites of limonene and perillyl alcohol.

The monoterpenes limonene and perillyl alcohol are effective therapeutic agents against advanced rat mammary cancer. Limonene is currently undergoing clinical testing in cancer patients. These monoterpenes and their oxygenated metabolites have been previously shown to inhibit protein prenylation in cultured cells. Since farnesylation of ras protein is critical for its ability to cause oncogenic transformation, inhibition of protein prenylation may be the basis of the anti-tumor effects of limonene and perillyl alcohol. In this study we test the ability of limonene and its oxygenated analogs to inhibit protein prenylation enzymes in vitro. Limonene and perillyl alcohol and their major in vivo metabolite, perillic acid, are weak inhibitors of both mammalian and yeast protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT). In contrast, a minor metabolite of both limonene and perillyl alcohol, perillic acid methyl ester, is a potent inhibitor of both enzymes. Perillic acid methyl ester is a competitive inhibitor of yeast PFT with respect to farnesyl pyrophosphate. These studies suggest that if the inhibition of protein prenylation is a mechanism for limonene's and perillyl alcohol's anti-cancer activities, these monoterpenes may be prodrugs that are converted into pharmacologically-active substances by metabolic modification.

Binding of prenylated and polybasic peptides to membranes: affinities and intervesicle exchange.

The small GTP-binding protein G25K and the protein K-Ras 4B contain prenyl groups (geranylgeranyl and farnesyl, respectively) that are thioether linked to a C-terminal cysteine which is methylated on its alpha-carboxyl group. These proteins, like many other prenyl proteins, also have a string of basic residues near their C-termini. A series of prenylated peptides based on the C-terminal sequences of human brain G25K and human K-Ras 4B were synthesized and analyzed for their membrane binding affinities. G25K peptides containing an N-terminal N-acetyltryptophan group were studied because their binding to membranes containing a trace of dansylated phospholipid could be detected by fluorescence resonance energy transfer. The G25K peptide lacking a prenyl group and a C-terminal methyl ester did not detectably bind to vesicles, and binding was enhanced by more than 500-fold if the peptide was geranylgeranylated. For the farnesylated peptide, methylation of the C-terminus increased membrane affinity by at least 60-fold if the vesicles contained phosphatidylserine and by 3-fold if they lacked this acidic lipid. The geranylgeranylated and methylated G25K peptide remains irreversibly attached to vesicles over several minutes only if the vesicles contain phosphatidylserine, whereas the corresponding nonmethylated or farnesylated and methylated peptides dissociate rapidly (less than a few seconds) from neutral or anionic vesicles. Farnesylation of the nonmethylated K-Ras 4B peptide enhances its affinity to vesicles containing acidic phospholipids (phosphatidylglycerol or phosphatidylserine) by 70-fold, and methylation leads to an additional dramatic (150-fold) increase in membrane affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity.

A protein geranylgeranyltransferase (PGT) that catalyzes the transfer of a 20-carbon prenyl group from geranylgeranyl pyrophosphate to a cysteine residue in protein and peptide acceptors was detected in bovine brain cytosol and partially purified. The enzyme was shown to be distinct from a previously characterized protein farnesyltransferase (PFT). The PGT selectively geranylgeranylated a synthetic peptide corresponding to the C terminus of the gamma 6 subunit of bovine brain G proteins, which have previously been shown to contain a 20-carbon prenyl modification. Likewise, a peptide corresponding to the C terminus of human lamin B, a known farnesylated protein, selectively served as a substrate for farnesylation by the PFT. However, with high concentrations of peptide acceptors, both prenyl transferases were able to use either peptide as substrates and the PGT was able to catalyze farnesyl transfer. Among the prenyl acceptors tested, peptides and proteins with leucine or phenylalanine at their C termini served as geranylgeranyl acceptors, whereas those with C-terminal serine were preferentially farnesylated. These results suggest that the C-terminal amino acid is an important structural determinant in controlling the specificity of protein prenylation.

Binding of the delta subunit to rod phosphodiesterase catalytic subunits requires methylated, prenylated C-termini of the catalytic subunits.

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.

Cloning and recombinant expression of a structurally novel human secreted phospholipase A2.

Mammals contain a diverse set of secreted phospholipases A(2) (sPLA(2)s) that liberate arachidonic acid from phospholipids for the production of eicosanoids and exert a variety of physiological and pathological effects. We report the cloning, recombinant expression, and kinetic properties of a novel human sPLA(2) that defines a new structural class of sPLA(2)s called group XII. The human group XII (hGXII) cDNA contains a putative signal peptide of 22 residues followed by a mature protein of 167 amino acids that displays homology to all known sPLA(2)s only over a short stretch of amino acids in the active site region. Northern blot and reverse transcription-polymerase chain reaction analyses show that the tissue distribution of hGXII is distinct from the other human sPLA(2)s with strong expression in heart, skeletal muscle, kidney, and pancreas and weaker expression in brain, liver, small intestine, lung, placenta, ovaries, testis, and prostate. Catalytically active hGXII was produced in Escherichia coli and shown to be Ca(2+)-dependent despite the fact that it is predicted to have an unusual Ca(2+)-binding loop. Similar to the previously characterized mouse group IIE sPLA(2)s, the specific activity of hGXII is low in comparison to that of other mammalian sPLA(2), suggesting that hGXII could have novel functions that are independent of its phospholipase A(2) activity.

Interfacial catalysis by phospholipase A2: evaluation of the interfacial rate constants by steady-state isotope effect studies.

The kinetics of hydrolysis of phospholipid vesicles by phospholipase A2 (PLA2) in the scooting mode can be described by the Michaelis-Menten formalism for the action of the enzyme in the interface (E*). E* + S in equilibrium E*S in equilibrium E*P in equilibrium E* + Products The values of the interfacial rate constants cannot be obtained by classical methods because the concentration of the substrate within the lipid bilayer is not easily manipulated. In the present study, carbonyl-carbon heavy atom isotope effects for the hydrolysis of phospholipids have been measured in both vesicles and in mixed micelles in which the phospholipid was present in the nonionic detergent Triton X-100. A large [14C]carbonyl carbon isotope effect of 1.12 +/- 0.02 was measured for the cobra venom PLA2-catalyzed hydrolysis of dipalmitoylphosphatidylcholine in Triton X-100. In contrast, no isotope effect (1.01 +/- 0.01) was measured for the action of the porcine pancreatic and cobra venom enzymes on vesicles of dimyristoylphosphatidylmethanol in the scooting mode. In a second experiment, the hydrolysis of vesicles was carried out in oxygen-18 enriched water. Analysis of the released fatty acid product by mass spectrometry showed that it contained only a single oxygen-18. All of these results were used to estimate both the forward and reverse commitments to catalysis. The lack of doubly labeled fatty acid demonstrated that the product is released from the E*P complex faster than the reverse of the esterolysis step. The small isotope effect in vesicles demonstrated that the E*S complex goes on to products faster than substrate is released from the enzyme. The relevance of these results to an understanding of substrate specificity and inhibition of PLA2 is discussed. In addition, the conditions placed on the values of the rate constants obtained in the present study together with results obtained in the other studies described in this series of papers have led to the evaluation of most of the interfacial rate constants for the hydrolysis of phospholipid vesicles by PLA2.

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.

Kinetic characterization of phospholipase A2 modified by manoalogue.

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of structural elements of bee venom phospholipase A2 involved in N-type receptor binding and neurotoxicity.

We have shown previously that neurotoxic venom secretory phospholipases A2 (sPLA2s) have specific receptors in brain membranes called N-type receptors that are likely to play a role in the molecular events leading to neurotoxicity of these proteins. The sPLA2 found in honey bee venom is neurotoxic and binds to this receptor with high affinity. In this paper, we have used a number of mutants of bee venom sPLA2 produced in Escherichia coli to determine the structural elements of this protein that are involved in its binding to N-type receptors. Mutations in the interfacial binding surface, in the Ca2+-binding loop and in the hydrophobic channel lead to a dramatic decrease in binding to N-type receptors, whereas mutations of surface residues localized in other parts of the sPLA2 structure do not significantly modify the binding properties. Neurotoxicity experiments show that mutants with low affinity for N-type receptors are devoid of neurotoxic properties, even though some of them retain high enzymatic activity. These results provide further evidence for the involvement of N-type receptors in neurotoxic processes associated with venom sPLA2s and identify the surface region surrounding the hydrophobic channel of bee venom sPLA2 as the N-type receptor recognition domain.

Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme.

Venom and mammalian secreted phospholipases A(2) (sPLA(2)s) have been associated with numerous physiological, pathological, and toxic processes. So far, structurally related group I and II sPLA(2)s have been found in vertebrates such as mammals and snakes, whereas group III sPLA(2)s have mainly been found in venom from invertebrates such as bees and scorpions. Here we report the cloning and expression of a cDNA coding for a human group III (hGIII) sPLA(2). The full-length cDNA codes for a signal peptide of 19 residues followed by a protein of 490 amino acids made up of a central sPLA(2) domain (141 residues) flanked by large N- and C-terminal regions (130 and 219 residues, respectively). The sPLA(2) domain is 31% identical to bee venom sPLA(2) and displays all of the features of group III sPLA(2)s including 10 cysteines. The hGIII sPLA(2) gene consists of at least 7 exons and maps to chromosome 22q. By Northern blot analysis, a 4.4-kilobase hGIII transcript was found in kidney, heart, liver, and skeletal muscle. Transfection of hGIII sPLA(2) cDNA in COS cells led to accumulation of sPLA(2) activity in the culture medium, indicating that the cDNA codes for a secreted enzyme. Using small unilamellar vesicles as substrate, hGIII sPLA(2) was found to be a Ca(2+)-dependent enzyme showing an 11-fold preference for phosphatidylglycerol over phosphatidylcholine and optimal activity at pH 8.

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2).

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.

The delta subunit of type 6 phosphodiesterase reduces light-induced cGMP hydrolysis in rod outer segments.

The delta subunit of the rod photoreceptor PDE has previously been shown to copurify with the soluble form of the enzyme and to solubilize the membrane-bound form (). To determine the physiological effect of the delta subunit on the light response of bovine rod outer segments, we measured the real time accumulation of the products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of delta subunit GST fusion protein (delta-GST) to this preparation caused a reduction in the maximal rate of cGMP hydrolysis in response to light. The maximal reduction of the light response was about 80%, and the half-maximal effect occurred at 385 nm delta subunit. Several experiments suggest that this effect was not due to the effects of delta-GST on transducin or rhodopsin kinase. Immunoblots demonstrated that exogenous delta-GST solubilized the majority of the PDE in ROS but did not affect the solubility of transducin. Therefore, changes in the solubility of transducin cannot account for the effects of delta-GST in the pH assay. The reduction in cGMP hydrolysis was independent of ATP, which indicates that it was not due to effects of delta-GST on rhodopsin kinase. In addition to the effect on cGMP hydrolysis, the delta-GST fusion protein slowed the turn-off of the system. This is probably due, at least in part, to an observed reduction in the GTPase rate of transducin in the presence of delta-GST. These results demonstrate that delta-GST can modify the activity of the phototransduction cascade in preparations of broken rod outer segments, probably due to a functional uncoupling of the transducin to PDE step of the signal transduction cascade and suggest that the delta subunit may play a similar role in the intact outer segment.

Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles.

The kinetics of hydrolysis of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles catalyzed by the high molecular weight phospholipase A2 from rat kidney show an anomalous behavior. The reaction progress lasts for several minutes and then stops after only 5-10% of the available substrate has been hydrolyzed. Addition of more enzyme but not more substrate leads to a new round of hydrolysis. Although this initially suggested that the enzyme becomes inactivated during the turnover, such a conclusion could not be substantiated. Addition of buffer containing 0.15 M NaCl and bovine serum albumin to the reaction after the progress ceased leads to the re-initiation of the lipolysis. The enzyme is not strongly inhibited by the reaction products. Although the enzyme does not bind irreversibly to vesicles composed of pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, it does become irreversibly trapped on vesicles that contain a critical mole percentage of reaction products. This trapping is the most likely explanation for the cessation of the reaction progress. Both the binding of enzyme to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles and the hydrolysis of 1-stearoyl-2-[3H]arachidonyl-sn-glycerophosphocholine contained in these vesicles require the presence of products. Furthermore, the trapping of enzyme is independent of catalytic turnover. The trapping is sensitive to the structure of the fatty acid present in the vesicles and requires the presence of divalent metals (either Ca2+, Sr2+, Ba2+, or Mg2+). Since the concentrations of the metals needed for the enzymatic activity correlate with the amounts needed to promote the trapping, it is suggested that the role of the metal is only to promote the interfacial binding of the enzyme.

On the diversity of secreted phospholipases A(2). Cloning, tissue distribution, and functional expression of two novel mouse group II enzymes.

Over the last decade, an expanding diversity of secreted phospholipases A(2) (sPLA(2)s) has been identified in mammals. Here, we report the cloning in mice of three additional sPLA(2)s called mouse group IIE (mGIIE), IIF (mGIIF), and X (mGX) sPLA(2)s, thus giving rise to eight distinct sPLA(2)s in this species. Both mGIIE and mGIIF sPLA(2)s contain the typical cysteines of group II sPLA(2)s, but have relatively low levels of identity (less than 51%) with other mouse sPLA(2)s, indicating that these enzymes are novel group II sPLA(2)s. However, a unique feature of mGIIF sPLA(2) is the presence of a C-terminal extension of 23 amino acids containing a single cysteine. mGX sPLA(2) has 72% identity with the previously cloned human group X (hGX) sPLA(2) and displays similar structural features, making it likely that mGX sPLA(2) is the ortholog of hGX sPLA(2). Genes for mGIIE and mGIIF sPLA(2)s are located on chromosome 4, and that of mGX sPLA(2) on chromosome 16. Northern and dot blot experiments with 22 tissues indicate that all eight mouse sPLA(2)s have different tissue distributions, suggesting specific functions for each. mGIIE sPLA(2) is highly expressed in uterus, and at lower levels in various other tissues. mGIIF sPLA(2) is strongly expressed during embryogenesis and in adult testis. mGX sPLA(2) is mostly expressed in adult testis and stomach. When the cDNAs for the eight mouse sPLA(2)s were transiently transfected in COS cells, sPLA(2) activity was found to accumulate in cell medium, indicating that each enzyme is secreted and catalytically active. Using COS cell medium as a source of enzymes, pH rate profile and phospholipid headgroup specificity of the novel sPLA(2)s were analyzed and compared with the other mouse sPLA(2)s.

Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2.

Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H(2)O(2) stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H(2)O(2) and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H(2)O(2), sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H(2)O(2) and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H(2)O(2) and sorbitol increased phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA(2) by H(2)O(2) occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA(2) activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.