Interactions of Platinum and Ruthenium Coordination Complexes with Pancreatic Phospholipase A2 and Phospholipids Investigated by MALDI TOF Mass Spectrometry

Phospholipase A₂ is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA₂ activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA₂ inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA₂ and phospholipids, as well as the application of MALDI‐TOF mass spectrometry for screening PLA₂ inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.     

Chronic Lithium Treatment Decreases Brain Phospholipase A2 Activity

Chronic lithium administration decreases the turnover of arachidonic acid (AA) in several brain phospholipids. This suggests that lithium may attenuate phospholipase A₂ (PLA₂) activity in brain. We now report effects of chronic lithium treatment on PLA₂ activity in postnuclear supernatant from rat brain: Enzyme activity was determined by two assay methods, radiometric and fluorometric, and measured the release of the fatty acid on the second acyl position (sn2) from choline and ethanolamine phospholipids. PLA₂ activity in brain postnuclear supernatant from rats chronically treated with lithium in the diet was significantly decreased (20–50%) when compared with controls. In vehicle or lithium-treated rats, PLA₂ activity was not significantly augmented or attenuated by the addition of calcium chelators, divalent cations or LiCl supplementation (1.0 mM) to postnuclear supernatant. These results suggest that a major therapeutic effect of lithium is to attenuate brain PLA₂ activity involved in signal transduction.     

Localizing the Membrane Binding Region of Group VIA Ca2+-independent Phospholipase A2 Using Peptide Amide Hydrogen/Deuterium Exchange Mass Spectrometry

The Group VIA-2 Ca²⁺-independent phospholipase A₂ (GVIA-2 iPLA₂) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA₂ in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA₂ as well as the interfacial lipid binding region.The Group VIA phospholipase A₂ is a member of the phospholipase A₂ superfamily that cleaves fatty acids from the sn-2 position of phospholipids (1, 2). The human Group VIA PLA₂³ gene yields multiple splice variants, including GVIA-1, GVIA-2, GVIA-3 PLA₂, GVIA Ankyrin-1, and GVIA Ankyrin-2 (3, 4). At least two isoforms, GVIA-1 and GVIA-2 iPLA₂, are active. Our laboratory purified and characterized the first mammalian iPLA₂, the 85-kDa GVIA-2 iPLA₂ (5), which became the first cloned iPLA₂ (6). This enzyme can hydrolyze the sn-2 fatty acyl bond of phospholipids and also has potent lysophospholipase and transacylase activity (7). GVIA iPLA₂ is involved in cell proliferation (8), apoptosis (9–11), bone formation (12), sperm development (13), and glucose-induced insulin secretion (14, 15), so its function may vary by cell and tissue.The human GVIA-2 iPLA₂ (806 amino acids), the form of the enzyme studied here, contains seven ankyrin repeats (residues 152–382), a linker region (residues 383–474) with the eighth repeat disrupted by a 54-amino acid insert (16), and a catalytic domain (residues 475–806). The active site serine of the GVIA iPLA₂ lies within a lipase consensus sequence (Gly-X-Ser⁵¹⁹-X-Gly) (1). The activity of GVIA iPLA₂ has been reported to be regulated through several mechanisms. A caspase-3 cleavage site at the N terminus of the enzyme has been identified that is clipped in vitro (17). This truncated form of the enzyme was hyperactive and reduced cell viability when overexpressed in HEK293 cells (17). Another possible control mechanism is through ATP binding on the ⁴⁸⁵GXGXXG motif (18).The activity of phospholipases depends critically on the interaction of the protein with phospholipid membranes. In vitro, GVIA iPLA₂ does not have any specificity for the fatty acid in the sn-2 position of substrate phospholipids (5). GVIA-2 iPLA₂ was found to be membrane-associated when overexpressed in COS-7 cells, and this was further confirmed in rat vascular smooth muscle cells (4, 19). The other active splice variant, GVIA-1, is cytosolic and not specific in targeting membrane surfaces (4, 19), indicating two different regulatory mechanisms between these two splice variants. The 54-residue insertion in the eighth ankyrin repeat alters the property of GVIA-2 iPLA₂ for membrane association. The ankyrin repeats have been reported to be involved in protein-protein interactions, such as 53BP2-p53, GA-binding protein α-GA-binding protein β, p16^INK4a-CDK6, and IκBα-NFκB (16). The ankyrin repeats of GVIA iPLA₂ may directly or indirectly assist membrane association because the catalytic domain by itself does not have activity (3). Determining the regions of the protein that interact with the membrane surface will allow for a more in-depth analysis of the regulatory mechanisms of the enzyme.There is an increasing interest in GVIA iPLA₂ because of its various newly discovered functions in vivo and in vitro. However, there is no published crystal or NMR structure to facilitate analysis on the molecular level. Amide hydrogen/deuterium exchange coupled with mass spectrometry (DXMS) has been widely used to analyze the interface of protein-protein interactions (20), protein conformational changes (21, 22), and protein dynamics (23), and we have now introduced it to study protein-phospholipid interactions (24, 25). There are also reports of using DXMS with homology modeling to validate enzymes where structural information does not exist (26). We used deuterium exchange along with homology modeling to generate models of the ankyrin repeats based on the Ankyrin-R (Protein Data Bank code 1N11) and of the catalytic domain based on patatin (Protein Data Bank code 1OXW). To study the interfacial activation of GVIA iPLA₂, we generated 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (PAPC) vesicles containing the methyl arachidonyl fluorophosphonate (MAFP) inhibitor, which binds to the active site and irreversibly inhibits GVIA iPLA₂ (7). By applying DXMS to the iPLA₂ and using our structural model, we were now able to monitor how GVIA iPLA₂ associates with phospholipid membranes.     

Lactadherin Inhibits Secretory Phospholipase A2 Activity on Pre-Apoptotic Leukemia Cells

Secretory phospholipase A₂ (sPLA₂) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA₂ concentrations are associated with autoimmune diseases and septic shock. Many sPLA₂’s do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A₂-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA₂ plateaued at 50–60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA₂ and >70% of human secretory phospholipase A₂-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA₂.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

A Role for Phospholipase A2 Activity in Membrane Tubule Formation and TGN Trafficking

We have investigated the role of phospholipase A₂ (PLA₂) enzymes in generating membrane tubules at the trans‐Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over‐expressing kinase dead protein kinase D were inhibited by the PLA₂ inhibitors ONO‐RS‐082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time‐lapse imaging revealed for the first time that PLA₂ antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA₂ enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking.     

Hydrolysis of Glycerophosphocholine Hydroperoxide by Phospholipase A2

In a sodium deoxycholate micellar solution with calcium chloride, a synthetic phospholipid hydroperoxide was hydrolyzed by porcine pancreatic phospholipase A₂ (PLA₂) and the reactivity observed for the hydroperoxide was demonstrated to be higher than the case with the unoxidized form. In these systems, it was found that the hydroperoxy group did not directly activate the PLA₂.     

Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA₁, GA₄, GA₉, GA₂₀). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (K _m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA₉ to GA₄ in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.     

Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP₂ binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP₂, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP₂-PLC interaction. This charge shielding of PIP₂ was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP₂ analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP₂ via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP₂ was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP₂ hydrolysis by PLC as triggered by M₁ muscarinic receptors with a potency order of Mg²⁺ < spermine⁴⁺ < neomycin⁶⁺. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg²⁺ and polyamines modulate the activity of PLCs by controlling the amount of free PIP₂ available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.     

Oligomerization Inhibits Legionella pneumophila PlaB Phospholipase A Activity

The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 μm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.     

Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A

An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K_m of the NADH oxidase after activation by lipids was four- to fivefold less than the K_m before activation. The V_max was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A₂ block the stimulation of the oxidase by auxins.     

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.     

Unimlecular Depurination of Substituted Deoxyadenosines by Fast Atom Bombardment Mass Spectrometry/Mass Spectrometry

Abstract

The effect of benzoyl substitution on the unimolecular depurination of deoxyadenosine, in the gas-phase, has been evaluated. The glycosidic bond dissociation of the conjugated acids and bases of the isomeric species occurs remote fran the charge sites.     

用于串联质谱鉴定多肽的计量方法

目前已有多种对串联质谱与数据库中多肽的理论质谱的一致性进行评估的高通量计量算法用于鸟枪法蛋白质组学 (shotgunproteomics)研究。然而这些方法操作时存在大量错误的多肽鉴定。这里提出一种新的串联质谱识别多肽序列的计量算法。该算法综合考虑了串联质谱中不同离子出现的概率、多肽的酶切位点数、理论离子与实验离子的匹配程度和匹配模式。对大容量的串联质谱数据集的测试表明 ,根据算法开发的软件PepSearch比目前最常用的软件SEQUEST有更好的鉴定准确性。PepSearch可从http : compbio.sibsnet.org projects pepsearch下载。     

Phospholipase A2 in cnidaria

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.     

磷脂酶A2的应用

磷脂酶Ａ2 (ｐｈｏｓｐｈｏｌｉｐａｓｅＡ2 ,ＰＬＡ2 ,ＥＣ 3 .1 .1 .4)即磷脂 2 酰基水解酶 ,是专一催化 3 Ｓｎ 磷酸甘油脂Ｃ 2位酯键的水解反应的酶 ,酶解产物为溶血磷脂和脂肪酸。ＰＬＡ2 不仅在生物体内具有很重要的生理功能 ,而且具有很高的应用价值 ,可广泛地应用在科学研究、磷脂改性、油脂精练、饲料添加剂、医疗等诸多方面。1 .用ＰＬＡ2 研究酶学、脂代谢和生物膜结构与功能ＰＬＡ2 (尤其是外分泌型的ＰＬＡ2 )的分子量较小 ,一般在 1 0～ 2 0ｋＤ之间 ,相对而言 ,结构较为简单。在蛇毒中 ,存在许多ＰＬＡ2 的同工酶 ,它们之…     

Phospholipase A(2)

Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.