Polarographic assay for phospholipase A2

A rapid, specific, and quantitative assay for phospholipase A₂ from Naja naja venom has been devised, in which phospholipid hydrolysis is measured as soybean lipoxidase-catalyzed oxygen incorporation into the ensuing unsaturated fatty acids. Under conditions where phospholipid was limiting, a linear relationship developed between the extent of oxygen uptake and the amount of egg lecithin metabolized. When phospholipase was rate limiting, the initial rate of oxygen consumption was a linear function of phospholipase concentration over a 14-fold range from 30 to 420 ng/ml. This linear relationship did not exist at higher phospholipase levels, probably due to the micellar nature of the phospholipid. Since this assay can readily detect as little as 17 ng/ml of phospholipase A₂ (Naja naja venom) and is insensitive to most potential interfering materials, it should be useful in a variety of applications.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Involvement of phospholipase A2 in neurodegeneration

Phospholipases A₂ are a heterogeneous class of enzymes that hydrolyse fatty acids from the sn-2 position of membrane phospholipids. Prolonged stimulation of phospholipase A₂ may damage membrane integrity, not only because of the loss of essential phospholipid from the lipid bilayer but also as a result of an uncontrollable Ca²⁺ influx. The increased levels of intracellular Ca²⁺ may be responsible for enhanced lipolysis, proteolysis and DNA fragmentation. This process along with the accumulation of lipid peroxidation products may be associated with neurodegeneration in acute neural trauma (ischemia, head and spinal cord injuries) and neurodegenerative diseases (Alzheimer's disease).     

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A₂ (PLA₂) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA₂-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA₂ enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA₂ enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA₂s), particularly the best studied enzyme Group IIA sPLA₂ in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA₂ in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA₂. Group VI calcium-independent PLA₂ enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA₂ enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.     

Inhibition of phospholipase A2 and phospholipase C by polyamines

The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A₂ (Naja naja) and phospholipase C (Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca²⁺, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.     

Interaction of phospholipase A2 and phospholipid bilayers

Binding of phospholipase A₂ from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC₁₄) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC₁₄ alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of the pig-pancreatic enzyme to vesicles of the diether-PC₁₄ could not be detected even after long incubation (up to 24 h) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

A chromogenic substrate for solid-phase detection of phospholipase A2

A method for solid-phase detection of phospholipase A₂ (PLA₂) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA₂. The substrate is hydrolyzed by PLA₂ into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA₂ spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA₂ were distinguishable from those producing an inactive mutant PLA₂, facilitating high-throughput screening in directed evolution of the enzyme.     

A rapid simple radiometric assay for phospholipase A2 activity

A rapid, simple radiochemical assay for phospholipase A₂ (PLA₂) activity is described in which incubations of homogenized tissue were directly applied to columns of silica gel absorbent. The metabolites and substrate were eluted from the columns with two different solvent systems so that each could be separated and quantified.     

Work in progress. Occurence of phospholipase A1 and A2 in human decidua

Phospholipase A₂, an enzyme which may regulate the formation of polyunsaturated fatty acids utilized for prostaglandin synthesis, was found to have significant higher activity in decidual than in myometrial tissue. The major part of phospholipase A₂ in the decidua had an acid pH optimum, which indicates that most of the enzyme is stored in the lysosomes of this tissue. These findings, together with previous observations, lend further support to the view that lysosomal phospholipase A₂ released within decidual cells might be a trigger of abortion and parturition.     

Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A₂ (PLA₂). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7–10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A₂ (PLA₂). Indeed, higher levels of calcium-independent PLA₂ activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA₂ activity is associated with increases in the expression of the 80-kDa calcium-independent PLA₂ (iPLA₂). Together, these data suggest that the 80-kDa iPLA₂ may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells.     

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography

Intracellular phospholipase A₂ was purified to homogenity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A₂ from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A₂ examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A₂ from other sources.     

Activation of phospholipase A2 by freshly added lysophospholipids

Reaction progress curves for the hydrolysis of dimyristoylphosphatidylcholine by pig pancreatic phospholipase A₂ exhibits a latency phase. Addition of 1-palmitoyllysophosphatidylcholine to the preformed vesicles reduces the latency phase and enhances the binding of phospholipase A₂ to the vesicles. In contrast, the binary codispersions prepared from diacylphospholipids premixed with lysophosphatidylcholine do not exhibit such enhanced susceptibility to the phospholipase. This effect appears to be due to organizational defects created by asymmetrical incorporation of lysophospholipid molecules into the outer monolayer of the vesicles, and the action of phospholipase is not observed when the additive is equilibrated in both the monolayers of the vesicles.     

Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A₂ hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA₂ shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA₂, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d₃₁-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA₂ molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA₂.     

Characterization of a rat kidney thromboxane A2 receptor: High affinity for the agonist ligand I-BOP

We have cloned a rat kidney thromboxane A2 receptor (TP) cDNA. This receptor was shown to be functional in that the thromboxane A₂ mimetics, U46619 and 1-BOP, elicited calcium transients in Xenopus oocytes and HEK293 cells expressing the TP receptor, respectively. Comparison of the affinities of the rat and human TP sites for the agonist radioligand [¹²⁵I]BOP showed that the rat TP site has about a ten-fold higher affinity for this drug (K_D = 0.5 vs. 4.4 nM) while the affinities of the two sites for other compound (U46619, I-PTH-OH) were the same. Our results are significant in that they identify a cloned TP as having a picomolar affinity for [¹²⁵I]BOP.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

Different forms of the cobalt-bleomycin A2 complex

Chromatographic analysis of Co-bleomycin on CM Sephadex C25 or silica shows that known components like bleomycin A₂, B₂, and A₁ can be separated into two forms. We observed an equilibrium between both forms and saw that the addition of certain salts influences the transition of one form into the other. A reaction scheme is suggested in which a different occupation of the sixth binding site of cobalt is correlated with the different forms observed. These different complexes may be an intramolecular adduct of the carbamoyl moiety of bleomycin, an external carboxylate adduct or an oxygen or hydroxyl adduct.