Cobra venom phospholipase

Selected entries from Methods in Enzymology [vol, page(s)] Cobra venom phospholipase A2 Naja naja naja, 197, 359 phospholipase A2 from rat liver mitochondria, 197, 365 assay and purification of phospholipase A2 from human synovial fluid in rheumatoid arthritis, 197, 373 purification of mammalian nonpan-creatic extracellular phospholipases A2, 197, 381 spleen phospholipases A2, 197, 390 purification and characterization of cytosolic phospholipase A2 activities from canine myocardium and sheep platelets, 197, 400. 

Tetrafluoroethene has been used as a 2-carbon difluoroacetic acid equivalent, methodology developed by Normanl and co-workers, in the synthesis of inhibitors of Cobra venom phospholipase A2.13 Conversion of the allyl alcohols into the 2,2-difluoropent-4-enoic acids 32 is performed in one pot. The crude acids 32 are then converted into the methyl esters 33. Although esters 33 can be obtained directly from the acyl fluorides 29, as originally described by Normant and co-workers,10 a two-step procedure facilitates the workup after the Claisen rearrangement. 

Kelley, M. J., Crowl, R. M., and Dennis, E. A. (1992). Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli. Biochim. Biophys. Acto 1118,107-115. 

Ortiz, A. R., Pisabarro, M. T., Gallego, J., and Gago, F. (1992). Implications of a consensus recognition site for phosphatidylcholine separate from the active site in cobra venom phospholipases As. Biochemistry 31,2887-2896. 

White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H., and Sigler, P. B. (1990). Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue. Science 250, 1560-1563. 

Similar inhibitory activity was observed for cobra venom phospholipase A2 and the mixture of bacterial alkylresorcinols in lecithin black lipid membrane and phospholipid emulsion systems. Almost complete inhibition (95%) of the enzyme studied was observed at a concentration of about 8 mM of resorcinolic lipids [351]. 

Fig. 2. Cobra venom phospholipase Aj (0.05/ig) was added to 1.4 ml of solution containing egg PC (2.6 mM) and egg PE (3.0 mM) in 48 mAf Triton X-100, 50 mM Tris-HQ, and 10 mil/Cha, pH 8.0, 40°C. P-NMR spectra are shown at the following times (min) after the initiation of reaction A, 0 B, 7 C, 27 D, 87. A JEOL PFT-100 NMR spectrometer Operating at 40.3 MHz was employed and spectra were recorded with H broadband decoupling. Peaks corresponding to lyso-PE (LPE), lyso-PC (LPC), PE, and PC are indicated. From Roberts et al. (1979). Copyright 1979 American Chemical Society. Note that the original figure has been altered to now show positive chemical shifts in the direction of decreasing field strength.

Fig. 13. (A) Action of cobra venom phospholipase Aj on mixed micelles consisting of 15 mM dipalmitoyl-PC (PC) and 120 mA/ Triton X-100. The enzyme concentration was 12.5 ng ml. The solution contained 10 mMCaCl2, 20% DjO, and SO mA/Tris-HO buffer, pH 6.0. (B) Action of Rhizopus arrhizus lipase (375 /ig ml ) under identical conditions. Spectra were obtained using the spectrometer described in Fig. 2. From Pliickthun and Dennis (1982a). Copyright 1982 American Chemical Society. Note that the original figure has been altered to now show positive chemical shifts in the direction of decreasing field strength.

A second group of myotoxic toxins, found almost exclusively in the venoms of cobras, are the cytotoxins (often called cobratoxins, cytolysins, cardiotoxins, or direct lytic factors). These, rather than phospholipases, are almost certainly the primary cause of muscle damage following bites by cobras. Their mechanism of action is not properly known, but it is certainly the case that their action is potentiated by the presence of phospholipases in the venom, even if the phospholipases concerned are not, themselves, myotoxic. The cytotoxins of cobra venom possess no hydrolytic activity of any kind. 

T6. The Action of Phospholipases The venom of the Eastern diamondback rattler and the Indian cobra contains phospholipase A2, which catalyzes the hydrolysis of fatty acids at the C-2 position of glycerophospholipids. The phospholipid breakdown product of this reaction is lysolecithin (lecithin is phosphatidylcholine). At high concentrations, this and other lysophospholipids act as detergents, dissolving the membranes of erythrocytes and lysing the cells. Extensive hemolysis may be life-threatening. 

R. Deems and Dennis. Phospholipase Ai horn cobra venom (Nqja nqja nftja). Methods Enzymoi. 77 703 (1931). 

R. Deems and E. Dennis. Phospholipase A2 from cobra venom (Naja naja naja). Methods Enzymol. 71 103 (1981). 

Synaptic vesicles, isolated from rat brain cortex, and cholinergic vesicles, isolated from the electric organ of Torpedo nobiliana, were broken down by a phospholipase A from cobra venom. The breakdown was accompanied by a release of acetylcholine. Morphological analysis revealed membrane fragments about one-third the size of the vesicle circumference. Subcellular fractions enriched in nerve ending membranes showed some phospholipase A activity. On the basis of these findings models of transmitter release, involving specific alterations of lipid components in the vesicular membrane, are discussed. 

Fig. 7. X-ray crystal structure of cobra venom (Naja naja naja) phospholipase with bound Ca showing a space-filling model of dimyristoyl phosphatidylethanolamine bound in the catalytic site. The ends of the fatty acid chains stick out of the enzyme and are presumably associated with the micelle or membrane (E.A. Dennis, 1994).

Snake venom is produced by the modified saliva glands of poisonous snakes. When a snake bites, venom is ejected through the fang of the snake. The venom of the eastern dia-mondback rattlesnake and the Indian cobra contains phospholipases, which are enzymes that catalyze the hydrolysis of the fatty acid on the center carbon of glycerophospholipids in the red blood cells. The resulting product, called lysophospholipid, causes breakdown of the red blood cell membranes. This makes them permeable to water, which causes hemolysis of the red blood cells. 

Thus, the original work established that the enzyme from Naja naja (Indian cobra) venom was a phospholipase A2. The situation then turned full circle by the discovery of another enzyme in rat liver which was a phospholipase Aj. 

In addition, it was desired to explore the effects that these small changes in intermolecular distance had on the enzymatic susceptibility of these lecithins to hydrolytic enzymes such as phospholipase A [3-5], a potent hydrolytic enzyme found in cobra venom. Thus, microgram quantities of enzyme were injected under this monolayer. By measming the rate of change of surface potential, one can indirectly measine the rate of reaction in the monolayer. It is assumed that these quantities [i.e., change in surface potential A(AV) and the extent of reaction] are proportional to each other. The kinetics of hydrolysis, as measured by a decrease in sinface potential, were studied for each lecithin monolayer as a fimction of initial surface pressure and are shown in Fig. 3 [6]. It was foimd that initially the reaction rate... 

The venoms of poisonous snakes contain (among other things) a class of enzymes known as phospholipases, enzymes that cause the breakdown of phospholipids. For example, the venoms of the eastern diamondback rattlesnake (Crotalus adamanteus) and the Indian cobra Naja naja) both contain phospholipase Ag, which catalyzes the hydrolysis of fatty acids at the C-2 position of glyc-erophospholipids. 

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. 

Tsai, l.-H., Wu, S. H., and Lo, T. B. (1981). The complete amino acid sequence of the phospholipase A2 from the venom of Naja naja atra (Taiwan cobra). Toxicon 19, 141-152. 

Group I secreted PLA. This group of phospholipases contains group lA enzymes such as those from cobra and krait venom, while the IB enzymes are the... 

The venoms of certain snakes contain enzymes called phospholipases that catalyze the hydrolysis of carboxylic ester bonds of phospholipids. The venom of the eastern diamondback rattlesnake (Crotalus adaman-teus) and that of the Indian cobra (Naja naja) both... 

Snake venoms a mixture of toxins produced in the venom glands (parotid gland, or salivary gland of the upper jaw) of venomous snakes (asps or hooded snakes, e.g. the cobra sea snakes vipers, e.g. puff adder, rattlesnake). They consist of highly toxic, antigenic polypeptides and proteins (which cause paralysis and death of the prey), and enzymes (which facilitate the spread of the toxins, and initiate digestion of undivided swallowed prey). The enzymes indude hya-luronidase (promotes spread of toxins), ATPase and acetylcholine esterase (paralysis), phospholipases (hemolysis), proteinases and L-amino add oxidases (tissue necrosis and blood clotting). 

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