Snake venom phospholipases

Hydrolytic enzymes phospholipases in snake venoms, endogenous... 

Harris, J.B. (1990). Phospholipases in snake venoms and their effects on nerve and muscle. In Snake Toxins (Harvey, A.L., ed.), pp. 91-129, Pergamon Press, Oxford. 

A. indica L. Indian Aristolochia, also known as Indian birthwort, ishvara (Sanskrit), or adagam (Tamil), is a bitter climber native to India. The medicinal material consists of the rhizome, which is to resolve inflammation (India), counteract insect poison, and as an antipyretic (Philippines and Vietnam). The rhizome contains aristolochic acid, which inhibits in vitro and dose-dependent phospholipid hydrolysis by the human synovial fluid phospholipase A2, snake venom phospholipase A2, porcine pancreatic phospholipase A2, and human platelet phospholipase A2 (2). 

Since predators of snakes (and humans) have to deal with snake venoms as defenses, they are included here, even though they serve in predation. Snake venoms are primarily enzymes (proteins), especially of the phospholipase A2 type, which breaks down cell membrane phospholipids hydrolytically. Other snake venoms such as cobrotoxin contain peptides with 60-70 amino acid residues. Pharmacologically, they have neurotoxic, cytotoxic, anticoagulant, and other effects. The neurotoxins, in turn, can have pre- or postsynaptic effects. Snake venoms with both neurotoxic and hemolytic effects on the heart are known as cardiotoxins. Cytotoxins attach to the cells of blood vessels and cause hemorrhage. Snake venom factors may stimulate or inhibit blood clotting. Finally, platelet-active factors can contribute to hemorrhage. 

Influence of Intermolecular Spacing on Enzymic Hydrolysis of Lecithin Monolayers. When snake venom phospholipase A is injected under a lecithin monolayer, it splits lecithin into lysolecithin and free fatty acid. The change in polar groups of the monolayer results in a change of surface potential. However, if prior to injection of enzyme into the subsolution, a lecithin monolayer is compressed to such a surface pressure that the active site of the enzyme is unable to penetrate the monolayer, hydrolysis does not proceed. For monolayers of dipalmitoyl, egg, soybean, and dioleoyl lecithins the threshold surface pressure values at which hydrolysis does not proceed are 20, 30, 37, and 45 dynes per cm., respectively (40). This is also the same order for area per molecule in their surface pressure-area curves, indicating that enzymic hydrolysis of lecithin monolayers is influenced by the unsaturation of the fatty acyl chains and hence the intermolecular spacing in monolayers (40). 

Lecithins and related phospholipids usually contain a saturated fatty acid in the C-l position but an unsaturated acid, which may contain from one to four double bonds, at C-2. Arachidonic acid is often present here. Hydrolysis of the ester linkage at C-2 yields a l-acyl-3-phosphoglycerol, better known as a Iysophosphatidylcholine. The name comes from the powerful detergent action of these substances which leads to lysis of cells. Some snake venoms contain phospholipases that form Iysophosphatidylcholine. Lysophosphatidic acid (l-acyl-glycerol-3-phosphate) is both an intermediate in phospholipid biosynthesis (Chapter 21) and also a signaling molecule released into the bloodstream by activated platelets.15... 

The phosphoric acid esters of diacyl glycerides, phospholipids, are important constituents of cellular membranes. Lecithins (phosphatidyl cholines) from egg white or soybeans are often added to foods as emulsifying agents or to modify flow characteristics and viscosity. Phospholipids have very low vapor pressures and decompose at elevated temperatures. The strategy for analysis involves preliminary isolation of the class, for example by TLC, followed by enzymatic hydrolysis, derivatization of the hydrolysis products, and then GC of the volatile derivatives. A number of phospholipases are known which are highly specific for particular positions on phospholipids. Phospholipase A2, usually isolated from snake venom, selectively hydrolyzes the 2-acyl ester linkage. The positions of attack for phospholipases A, C, and D are summarized on Figure 9.7 (24). Appropriate use of phospholipases followed by GC can thus be used to determine the composition of phospholipids. 

I various subcellular locations within eukaryotic cells. Some of these enzymes are specific for particular polar head-groups others are nonspecific. Phospholipase A2 is a major component of snake venom (cobra and rattlesnake) and is partially involved in the deadly effects of these venoms. Because of the high concentration of phospholipase A2 in these venoms, this enzyme has been studied intensively. The pancreas is also rich in phospholipase A2, which is secreted into the intestine for digestion of dietary phospholipids. 

Snake venoms contain many types of lipases, including phospholipase A2. Why do small amounts of this enzyme contribute to some of the toxic effects of snake venom (Bee venom contains a protein that stimulates phospholipase A2.)... 

Snake venoms have been studied extensively their effects are due, in general, to toxins that are peptides with 60 to 70 amino acids. These toxins are cardiotoxic or neurotoxic, and their effects are usually accentuated by the phospholipases, peptidases, proteases, and other enzymes present in venoms. These enzymes may affect the bloodclotting mechanisms and damage blood vessels. Snake bites are responsible for less than 10 deaths per year in the United States but many thousand worldwide. 

Hanna PA, Jankovic J, Vincent A (1999) Comparison of mouse bioassay and immunoprecipitation assay for botulinum toxin antibodies. J Neurol Neurosurg Psychiatry 66 612-16 Hanson MA, Stevens RC (2000) Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 A resolution. Nat Struct Biol 7 687-92 Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001) The architecture of active zone material at the frog s neuromuscular junction. Nature 409 479-84 Harris JB (1997) Toxic phospholipases in snake venom an introductory review. Symp. zool. Soc. Lond. 70 235-50... 

Kini RM (1997) Venom phospholipase a2 enzymes. John Wiley Sons, Chichester Kini RM, Evans HJ (1989) A model to explain the pharmacological effects of snake venom phospholipases a2. Toxicon 27 613-35... 

Lee CY, Chang CC, Chen YM (1972) Reversibility of neuromuscular blockade by neurotoxins from elapid and sea snake venoms. Taiwan Yi Xue Hui Za Zhi 71 344-9 Lee CY, Tsai MC, Chen YM, Ritonja A, Gubensek F (1984) Mode of neuromuscular blocking action of toxic phospholipases A2 from vipera ammodytes venom. Arch Int Pharmacodyn Ther 268 313-24... 

The phospholipases A2 isolated from snake venom appear able to attack equally well phosphatidylcholine, phosphatidylethanolamine, and other phosphoglycerides, if these compounds are presented in the proper physical configuration. As noted above, the latter can be accomplished through the use of a diethyl ether-methanol-water mixture or by inclusion of a suitable detergent. 

Partial (Semi) Synthesis. A blend of two methodologies can be used quite effectively to prepare mixed acid phosphatidylcholines, which are of prime importance in elucidating the structure of naturally occurring phospho-glyceride and phospholipase A2 activity. Perhaps the best scenario would be to illustrate the activity of the phospholipase A2 (from Crotalus adamanteus snake venom) toward a racemic phosphatidylcholine sample and toward individual sn-1 and sn-3 enantiomers. In each case the same result would be found, and so only the racemic mixture reaction is depicted in Figure 4-8. 

In the biochemical method, the enzyme phospholipase A2, isolated from Naja naja snake venom can attack the native alkenylacylglycerophosphocho-line and liberate completely the esterified fatty acid and the alkenyl(lyso)glyc-erophosphocholine. On the basis of the stereospecific mode of attack of this enzyme on the 2-acyl ester position of sn-3 phosphoglycerides, it can be concluded that the naturally occurring alkenylacylglycerophosphocholine possessed the sn-3 stereochemical configuration. 

Phospholipase A2 Action. Incubation of phosphatidylserine with phospholipase A2 obtained from Crotalus adamanteus or Naja Naja snake venom will show that the serine-containing phosphoglyceride was smoothly and completely converted to a lysophosphatidylserine with liberation of 1 mol of fatty acid per mole of lipid P. The experimental procedure was the same as the one described before in this and in the previous chapter. The products of the reaction can be recovered by thin-layer chromatography on Whatman K6 plates in a solvent system of chloroform-acetone-methanol-acetic acid-water (4.5 2 1 1.3 0.5, v/v). 

Phospholipase A2 (snake venom) will attack phosphatidylglycerol smoothly and to completion under conditions previously described in chapter 4. One mole of fatty acid is released per mole of lipid phosphorus. This result supports an sn-3 configuration for the phosphatidic acid portion of the molecule. 

Phospholipase A2 isolated from snake venom (Crotalus adamanteus) can attack cardiolipin with liberation of 2 mol of fatty acid per mol of phosphorus. This result argues for an sn-3 configuration for the native cardiolipin molecule. 

Teshima, K., Kitagawa, Y., Samejima, Y., Kawauchi, S., Fijii, S., Ikeda, K., Hayashi, K., and Omori-Satoh, T. (1989). Role of calcium in the substrate binding and catalytic functions of snake venom phospholipases A. J. Biochem. (Tokyo) 106, 518-527. 

Tsai, l.-H., Liu, H.-C., and Chang, T. (1987). Toxicity domain in presynaptically toxic phospholipase A2 of snake venom. Biochim. Biophys. Acta 916,94-99. 

Presynaptic toxins - polypeptide snake venoms This group contains multimeric polypeptides containing subunits with phospholipase Aj activity, and often contains other subunits with a chaperone role. Toxicity does not normally rest on enzyme activity alone, and binding may occur other than on neuronal tissue, e.g. some cause skeletal muscle cytotoxicity. Examples from snake and viper venoms include agkistrodotoxin, ammodytoxin A. P-bungarotoxins. mojave toxin, notexin. taipoxin and textilotoxin. 

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