Neuronal receptors for phospholipases A(2 )and beta-neurotoxicity

Some phospholipases A(2) interrupt neuromuscular communication by blocking the release of neurotransmitter into the synaptic cleft. Despite numerous studies, the molecular mechanism of their action is still largely obscure. In this review the best-characterized receptors for beta-neurotoxins are presented. We propose a model which could be useful in investigating the apparent inconsistency between the observed heterogeneity in the neuronal binding of beta-neurotoxins and the very similar pathomorphological and electrophysiological effects which they produce in the intoxicated tissue. We assume that beta-neurotoxins enter the nerve ending to exert their toxic effect. The model involves different pathways for phospholipase A(2) neurotoxins to reach the site of action inside the neuron, their respective extra- and intracellular neuronal receptors being key features of the pathway. Once in the nerve cell, beta-neurotoxins impair the function of the synaptic vesicles by phospholipid hydrolysis of the inner leaflet of the vesicle bilayer. The proportion of the products of the phospholipid hydrolysis, lysophospholipids and phospholipids in the membrane, has been demonstrated to be very important for the shaping of the membrane, affecting its fusogenic properties. Due to the same final step in the action of beta-neurotoxins, phospholipid hydrolysis, the consequences of their poisoning are practically identical.     

Characterization of type 2 opiate receptors

Recent evidence suggests that the Type 1 opiate receptor (in rat striatal patches) is a mobile receptor which is able to adopt a mu, delta, or kappa opiate receptor ligand selectivity pattern under appropriate conditions. In this paper, we have investigated such a possibility for Type 2 opiate receptors which are visualized diffusely over rat striatum. Ligand selectivity analysis suggested that the Type 2 opiate binding site is equivalent to a delta opiate receptor. The auto-radiographic distribution of Type 2 opiate binding sites is diffuse over most areas of rat brain. Thus, Type 2 opiate binding sites are different from Type 1 opiate receptors which are very discretely distributed in rat brain. Our results suggest that Type 2 opiate receptors, unlike Type 1 opiate receptors, are receptors locked in a delta-like ligand selectivity conformation.     

Properties of receptors for neurotoxic phospholipases A2 in different tissues

A radioiodinated derivative of OS₂ (¹²⁵I–OS₂), a neurotoxic monochain phospholipase A₂ isolated from taipan venom, was previously found to bind to a specific brain membrane receptor with very high affinity.¹²⁵I–OS₂ is now used to identify the properties of neurotoxic phospholipase receptors in other tissues. Heart, skeletal muscle, kidney, lung, liver, pancreas, and smooth muscle membranes also contain high-affinity binding sites for toxic phospholipases A₂. In most tissues, two different types of receptor sites have been characterized for¹²⁵I–OS₂ with K_d1 and K_d2 values in the 1–5 pM and the 10–50 pM range respectively. Whereas all receptors are similar in the different tissues in terms of their affinity for¹²⁵I–OS₂, maximal binding site capacities were very different, varying from 1.4 pmol/mg of protein in brain to 0.01 pmol/mg of protein in pancreaas. In brain, heart, and skeletal muscle, receptor densities vary with in vivo development. Affinity labeling experiments have identified the subunit composition of OS₂ receptors and indicated that these receptors do not have identical structures in the different tissues. Binding competition studies with OS₂ and other toxic phospholipases showed tissue-dependent pharmacological profiles. All these results taken together suggest the existence of a family of receptors for neurotoxic phospholipases.The abbreviations used are PLA₂ phospholipase A₂ - DSS suberic acid bis-N-hydroxysuccinimide ester - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - OS₁ Oxyuranus scutellatus scutellatus toxin 1 - OS₂ Oxyuranus scutellatus scutellatus toxin 2 Special issue dedicated to Dr. Lawrence Austin.     

Membrane protein solubilization: recent advances and challenges in solubilization of serotonin1A receptors

Solubilization of integral membrane proteins is a process in which the proteins and lipids that are held together in native membranes are suitably dissociated in a buffered detergent solution. The controlled dissociation of the membrane results in formation of small protein and lipid clusters that remain dissolved in the aqueous solution. Effective solubilization and purification of membrane proteins, especially heterologously-expressed proteins in mammalian cells in culture, in functionally active forms represent important steps in understanding structure-function relationship of membrane proteins. In this review, critical factors determining functional solubilization of membrane proteins are highlighted with the solubilization of the serotonin 1A receptor taken as a specific example.     

Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins

Secreted phospholipases A(2) (sPLA(2)s) form a large family of structurally related enzymes which are widespread in nature. Snake venoms are known for decades to contain a tremendous molecular diversity of sPLA(2)s which can exert a myriad of toxic and pharmacological effects. Recent studies indicate that mammalian cells also express a variety of sPLA(2)s with ten distinct members identified so far, in addition to the various other intracellular PLA(2)s. Furthermore, scanning of nucleic acid databases fueled by the different genome projects indicates that several sPLA(2)s are also present in invertebrate animals like Drosophila melanogaster as well as in plants. All of these sPLA(2)s catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids, and thus could be important for the biosynthesis of biologically active lipid mediators. However, the recent identification of a variety of membrane and soluble proteins that bind to sPLA(2)s suggests that the sPLA(2) enzymes could also function as high affinity ligands. So far, most of the binding data have been accumulated with venom sPLA(2)s and group IB and IIA mammalian sPLA(2)s. Collectively, venom sPLA(2)s have been shown to bind to membrane and soluble mammalian proteins of the C-type lectin superfamily (M-type sPLA(2) receptor and lung surfactant proteins), to pentraxin and reticulocalbin proteins, to factor Xa and to N-type receptors. Venom sPLA(2)s also associate with three distinct types of sPLA(2) inhibitors purified from snake serum that belong to the C-type lectin superfamily, to the three-finger protein superfamily and to proteins containing leucine-rich repeats. On the other hand, mammalian group IB and IIA sPLA(2)s can bind to the M-type receptor, and group IIA sPLA(2)s can associate with lung surfactant proteins, factor Xa and proteoglycans including glypican and decorin, a mammalian protein containing a leucine-rich repeat.     

Inactivation of hepatic glucocorticoid receptors by heparin

Heparin dramatically enhanced the rate of unbound glucocorticoid receptor inactivation in vitro in a concentration, time and temperature-dependent manner. Control specific binding decreased only about 25% after incubation for 6 h at 4°C. However in the presence of heparin (40 μg per ml cytosol) receptor binding decreased about 75%. At 25°C liver receptor specific binding was found to have a half0life of about 60 min in control cytosol. However, in the presence of heparin (40 μg per ml cytosol) the glucocorticoid receptor had a half-life of only 15 min at 25°C. Interestingly, 10 mM molybdate (with or without 5 mM dithiothreitol) greatly inhibited heparin-dependent receptor inactivation at 4°C. Dithiothreitol (alone) significantly stabilized receptor binding in control samples at 4°C, but provided no protection from heparin-dependent receptor inactivation. Heparin had no apparent inactivating effect on prebound glucocorticoid receptor complexes at 4°C. Interestingly however, heparin altered the sedimentation coefficient of prebound hepatic glucococorticoid-receptor complexes in low salt gradients from 7–8 S to about 3–4 S. When molybdate plus dithiothreitol were added with heparin, the sedimentation coefficient was found to be approx. 6—7 S. These results demonstrate that heparin, which is often used pharmacologically and which occurs naturally in animal tissues, has significant effects on liver glucocorticoid receptors in vitro.     

Activation of human inflammatory cells by secreted phospholipases A2

Secreted phospholipases A(2) (sPLA(2)s) are enzymes detected in serum and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Different isoforms of sPLA(2)s are expressed and released by human inflammatory cells, such as neutrophils, eosinophils, T cells, monocytes, macrophages and mast cells. sPLA(2)s generate arachidonic acid and lysophospholipids thus contributing to the production of bioactive lipid mediators in inflammatory cells. However, sPLA(2)s also activate human inflammatory cells by mechanisms unrelated to their enzymatic activity. Several human and non-human sPLA(2)s induce degranulation of mast cells, neutrophils and eosinophils and activate exocytosis in macrophages. In addition some, but not all, sPLA(2) isoforms promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes and endothelial cells. These effects are primarily mediated by binding of sPLA(2)s to specific membrane targets (heparan sulfate proteoglycans, M-type, N-type or mannose receptors) expressed on effector cells. Thus, sPLA(2)s may play an important role in the initiation and amplification of inflammatory reactions by at least two mechanisms: production of lipid mediators and direct activation of inflammatory cells. Selective inhibitors of sPLA(2)-enzymatic activity and specific antagonists of sPLA(2) receptors are current being tested for pharmacological treatment of inflammatory and autoimmune diseases.     

Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A2

In an effort to identify the anticoagulant region of venom phospholipases A2, we have systematically compared the amino acid sequences of strong, weak and non-anticoagulant phospholipases. The comparison disclosed several significant substitutions in the region between residues 54 and 77 (homology numbers). This proposed anticoagulant region is positively charged in strong, but negatively charged in weak and non-anticoagulant phospholipases. The microenvironment of a tryptophan residue falls within the proposed region, accounting for the differential characteristics of intrinsic fluorescence changes observed at 335 nm after the binding of phospholipid vesicles to strong and weak anticoagulants. Four lysine residues are located in specific positions in the "anticoagulant" region of strong anticoagulants, and should form a cationic surface, based on analogy with the available crystallographic structures. The chemical modification of lysine, arginine, tyrosine, and tryptophan residues and carboxylate groups, performed by other investigators, not only provides added support for the predicted site, but also confirms the essentiality of the positive charges in the site. This region may participate in the formation of a specific preferential hydrolytic complex leading to the strong anticoagulant effect. The anticoagulant region is distinct and separate from the predicted neurotoxic and myotoxic sites, and is located on the opposite surface of the phospholipase molecule.     

The non-venom insect phospholipases A2

Phospholipases A(2) (PLA(2)s) are responsible for releasing the fatty acid moiety from the sn-2 position of phospholipids. These enzymes are virtually ubiquitous proteins known from all major biological taxa. Various PLA(2)s act in a wide array of biological processes, including digestion of dietary lipids, cellular homeostasis, intra- and intercellular signaling, host defense and at least a few ecological interactions. PLA(2) activities have been recorded in a small number of insect species, which can be taken to represent the broad group, Insecta. Within insects, PLA(2)s act in functions expected from the background on these enzymes. So far, we know PLA(2)s act in lipid digestion, cellular host defense signaling, reproduction and in organismal-level metabolism. Additional PLA(2) actions are certain to emerge. This is the first article devoted to assembling the known information on insect PLA(2)s. I review the scant information available on the biological actions of PLA(2)s in insects, relate new findings on insect pathogens that disrupt insect immune functions by inhibiting PLA(2)s and mention the few reports of sequence information on insect PLA(2)s. Finally, I offer a brief prospectus on future research into insect PLA(2)s. There are two overarching points in this paper. One, there remains a great deal to learn about insect PLA(2)s and two, some of the findings on insect PLA(2)s will have meaningful practical significance.     

Interfacial enzymology of parvovirus phospholipases A2

The capsid of parvoviruses proteins were recently shown to contain secreted phospholipase A(2) (sPLA(2))-like activity that is required during host cell entry. Parvoviral PLA(2) domains have little sequence identity with sPLA(2)s and lack disulfide bonds. In the present study, after bacterial expression and purification, the biochemical characterizations of these first PLA(2)s identified in viruses have been investigated, and a comparison has been made with other known PLA(2)s. The specific activities of three viral PLA(2)s differed by 3 orders of magnitude, with porcine parvovirus PLA(2) displaying a specific activity similar to that of the most active sPLA(2)s (e.g. human group IIA) and the human AAV2 and B19 parvoviral enzymes displaying approximately 10(3) lower specific activities (similar to human sPLA(2) groups IIE and XIIA). These differences were not caused by weaker Ca(2+) or interfacial binding. The specific activities of the viral PLA(2)s on zwitterionic or anionic phospholipid vesicles were comparable. The viral PLA(2)s did not display a preference for unsaturated versus saturated sn-2 fatty acyl chains and hydrolyzed all major classes of glycero-phospholipids except phosphatidylinositol. Incubation of mammalian cells with porcine parvovirus PLA(2) led to the release of arachidonic acid into the culture medium. Interestingly, among nine previously known sPLA(2) inhibitors, only a subset showed inhibition of the viral PLA(2)s and with weak potency, indicating that the active sites of these new enzymes are structurally distinct from those of sPLA(2)s. Based on these distinct enzymatic and structural properties, we propose to classify the parvovirus PLA(2)s within the PLA(2) superfamily as group XIII enzymes.     

Molecular dynamics simulations of phospholipases A2

An extensive molecular dynamics study of phospholipases A2 from pancreatic bovine and Crotalus atrox venom has shown that the well-conserved homologous core of the phospholipases A2, including the so called catalytic network, is very stable during the course of the calculations. The fluctuations which occur are located in segments which have significantly different three-dimensional conformations in the two phospholipases A2 studied, suggesting that a particularly stable core conformation gives rise to a large homologous family of similar three-dimensional structure. The calcium ion, which exhibits a crucial structural role in the monomeric phospholipases A2, appears not to be required to stabilize the C.atrox dimer. Moreover, the behaviour of the dimeric structure during the dynamics raises the question of a possible dissociation of the two subunits into functional monomers.     

Electroacupuncture analgesia is mediated by stereospecific opiate receptors and is reversed by antagonists of type I receptors

Dextronaloxone, a recently synthesized stereoisomer, which was shown to possess much less opiate receptor affinity than levonaloxone, produces no reversal of electroacupuncture analgesia (EAA) in mice. Since levonaloxone completely reverses EAA, this proves that stereospecific opiate receptors are involved. It has been reported that there are two classes of opiate receptors: Type I and Type II. Type I opiate receptors may be responsible for opiate analgesia. Antagonists of Type I receptors, levonaloxone, naltrexone, cyclazocine and diprenorphine, all block electroacupuncture analgesia at low doses. All together, these results strongly support the hypothesis that electroacupuncture analgesia is mediated by opiate receptors. Possibly Type I receptors are the major component of this system.     

Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.     

Differential solubilization of nuclear glucocorticoid receptors by DNAase I and DNAase II

This study analyzes the sensitivity of nuclear bound glucocorticoid receptors to solubilization from nuclei by DNAase I and DNAase II. Thymocytes were incubated with 10(-8) M [3H]dexamethasone, [3H]cortisol or [3H]triamcinolone acetonide, without or with 10(-6) M unlabelled dexamethasone, for 30 min at 37 degrees C and nuclei from these cells were digested with either DNAase I and DNAase II. DNAase I for 2 h at 3 degrees C leads to solubilization of 60% of the nuclear DNA and release of 10--20% triamcinolone acetonide-receptor, 30--40% dexamethasone-receptor and 85--90% cortisol-receptor. DNAase II at the same enzymatic concentration solubilizes only 10--20% of the nuclear DNA, but releases 40--50% triamcinolone-receptor, 60--70% dexamethasone-receptor and 100% cortisol-receptor. Release of nuclear bound dexamethasone-receptor by DNAase I parallels the solubilization of DNA, reaching maximum values by 2 h at 3 degrees C, whereas maximal release by DNAase II is obtained within 45 min when DNA solubilization is not complete. When nuclei initially extracted with DNAase I are re-extracted with DNAase II, greater than 65% of the DNAase I residual dexamethasone-receptors are solubilized, whereas DNAase I is ineffective in solubilizing DNAase II residual dexamethasone-receptors. DNAase I solubilizes only 30% of the 0.4 M KCl residual dexamethasone-receptor whereas DNAase II digests over 90% of this fraction. DNAase I extracts of nuclear dexamethasone-receptor chromatograph on G-100 Sephadex as a single radioactive peak just after the void volume, whereas DNAase II extracts of nuclear dexamethasone-receptor chromatograph as two peaks of radioactivity, one which is similar to the DNAase I solubilized receptor and a second broad peak of macromolecular bound radioactivity which is smaller in size.     

The molecular structure of opiate receptors

Examples are given which demonstrate that the opiate receptor can be separted from and subtypes by their physical parameters. When the subunit composition of the subtypes are compared, no definite differences are encountered. The data from the literature are also contradictory. This may in part be explained by the fact that the different receptors appear to contain a structurally common high affinity binding site. A possible hypothesis would be that the subtypes differ from each other by the number of subunits.Abbreviations CHAPS 3/cholamidopropyl-dimethylammonio 1-propansulfonate - DAGO D-Ala ²-Me-Phe⁴-Gly-ol⁵-enkephalin - DALA d-Ala²-Leu⁵-enkephalin - DALECK d-Ala²-Leu⁵-enkaphalin chloromethyl ketone - EGF Epidermal growth factor - EKC ethylketocyclazocine - SDS-PAGE Sodiumdodecylsulfate polyacrylamide gel elecctrophoresis Special Issue Dedicated to Dr. Abel Lajtha.     

Control of free arachidonic acid levels by phospholipases A2 and lysophospholipid acyltransferases

Arachidonic acid (AA) and its oxygenated derivatives, collectively known as the eicosanoids, are key mediators of a wide variety of physiological and pathophysiological states. AA, obtained from the diet or synthesized from linoleic acid, is rapidly incorporated into cellular phospholipids by the concerted action of arachidonoyl-CoA synthetase and lysophospholipid acyltransferases. Under the appropriate conditions, AA is liberated from its phospholipid storage sites by the action of one or various phospholipase A₂ enzymes. Thus, cellular availability of AA, and hence the amount of eicosanoids produced, depends on an exquisite balance between phospholipid reacylation and hydrolysis reactions. This review focuses on the enzyme families that are involved in these reactions in resting and stimulated cells.