Phospholipase

Several studies suggest that cGMP and PKG inhibit agonist-evoked phospholipase C formation (Rap-oport, 1986 Takai et al., 1981 Hirata et al., 1990). Ruth et al. (1993) reported that CHO cells overexpressing PKG demonstrated an attenuated thrombin-stimulated IP3 response in the presence of cGMP. Control cells not expressing PKG were insensitive to the 

FIGURE 4 Complexity of actions of PKG in various smooth muscle cells. PKG may inhibit phospholipase C (PLC) activation or IP3 receptor gating to inhibit the mobilization of [Ca +l, within the cell. PKG may also stimulate Ca + removal from the cytoplasm by activating BK channels or stimulating Ca + ATPase activity through the phosphorylation of phospholamban (PLB). Other modes of action not depicted in the model include inhibition of contractile protein function, inhibition of L-type channels, and regulation of cytoskeletal events. 

Another large class of effector molecules that are activated by G-proteins are the phospholipases of type C. 

Phospholipases are enzymes that cleave phospholipids. Phosphohpases of type Al, A2, C and D are differentiated according to the specificity of the attack point on the phospholipid. The bonds cleaved by these phospholipases are shown in Fig. 5.24a. 

Cleavage of inositol-containing phospholipids by phospholipase C is of particular regulatory importance. Phospholipase C catalyzes the release of diacyl glycerol and inositol-l,4,5-triphosphate from phosphatidyl inositol-4,5-diphosphate, a phospholipid 

Phosphohpases of type CP and Cy are activated via central signaling pathways  

Phospholipases of type CP are activated by Gq proteins which conmumicate themselves with various 7-hehx transmembrane receptors. The initiating external signals are diverse (see Fig. 5.14) and include hormones, neurohormones and sensory signals such as odorous agents and light (in non-vertebrates). 

Although the phospholipases of type Cfi and Cy catalyze the same biochemical reaction, they are activated via different signaling pathways. The Cfi subfamily participates in G protein signaling while the members of the Cy subfamily function as effectors of receptor tyrosine kinases (see Chapter 8). 

The effector function of phospholipase Cfi enzymes in G protein signaling is based on and mediated by the following functions and interactions  

Encompassing approx 6000 medicinal plant species, the medicinal flora of Asia and the Pacific comprise a fantastic source of pharmacologically active products, and the number of plant species principally used for the treatment of inflammation can be estimated to be more that 380. This chapter will focus on the potentials of medicinal plants of Asia as a source of original anti-inflammatory drugs, with particular interest payed to inhibitors of phospholipase A2, COX, lipoxygenases, elastase, and NOS. 

Other medicinal features to consider when searching for plants with potential as phospholipases A2 are abortifacient, analgesic, antipyretic, and hypoglycemic uses. Such features are present in the following plant species. 

A review summarizing the classical work on phosphoHpases is that of Ercoli (1940). More recent reviews have been prepared by Rossiter (1960), Kates (1960) and An SELL and Hawthorne (1964). 

The enzyme has a very wide distribution, being present in snake venoms, animal tissues, plants, bacteria and fungi. Venom phosphoHpase A was crystallized by De (1944). 

A finding of interest is the discovery of Hanahan (1952) that the activity of phosphoHpase A is increased by the addition of diethyl ether, a property the enzyme shares with other phosphoHpases. Dawson (1963) concluded that the stimulatory effect of ether on the hydrolysis of lecithin by phosphoHpase A is the result of penetration of ether molecules into the Hpid miceUe. Such a penetration causes a wider spacing of the molecules of lecithin orientated in the Hpid-water interface, aUowing more ready access of the enzyme to the susceptible acyl ester bond. Also the fatty acids Hberated in the enzymic reaction will be more readily removed from the surface of the lecithin miceUes. Such fatty acids will be replaced by fresh substrate molecules and so will not impede the action of the enzyme. It should also be pointed out that phosphoHpids oriented in a Hpoprotein complex are more readily hydrolysed by phosphoHpase A than is a suspension of purified lecithin (CoNDREA et al. 1963). 

PhosphoHpase A, which is inactive towards lysolecithin (Fairbairn 1945), is capable of removing both saturated and unsaturated fatty acids from lecithins, but at different rates (Hanahan, Rodbell and Turner 1954, Long and Penny 1957). Long and Penny (1957) showed that the enzyme can remove fatty acid from both natural and synthetic L-a-lecithins, but not from D-a-lecithins. The effect of the enzyme on a series of synthetic substrates was investigated by van Deenen and de Haas (1963). 

There is now considerable evidence that it is the fatty acid esterified in the /S-position that is selectively detached with the production of cx -acyl-a-glycerol-phosphorylcholine (VI) (Tattrie 1959, Hanahan, Brockerhoff and Barron 

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Hydrolases. Enzymes catalysing the hydrolytic cleavage ofC —O, C —N and C —C bonds. The systematic name always includes hydrolase but the recommended name is often formed by the addition of ase to the substrate. Examples are esterases, glucosidases, peptidases, proteinases, phospholipases. Other bonds may be cleaved besides those cited, e.g. during the action of sulphatases and phosphatases. 

Although extraction of lipids from membranes can be induced in atomic force apparatus (Leckband et al., 1994) and biomembrane force probe (Evans et al., 1991) experiments, spontaneous dissociation of a lipid from a membrane occurs very rarely because it involves an energy barrier of about 20 kcal/mol (Cevc and Marsh, 1987). However, lipids are known to be extracted from membranes by various enzymes. One such enzyme is phospholipase A2 (PLA2), which complexes with membrane surfaces, destabilizes a phospholipid, extracts it from the membrane, and catalyzes the hydrolysis reaction of the srir2-acyl chain of the lipid, producing lysophospholipids and fatty acids (Slotboom et al., 1982 Dennis, 1983 Jain et al., 1995). SMD simulations were employed to investigate the extraction of a lipid molecule from a DLPE monolayer by human synovial PLA2 (see Eig. 6b), and to compare this process to the extraction of a lipid from a lipid monolayer into the aqueous phase (Stepaniants et al., 1997). 

Ferguson, K.M., et al. Structure of the high affinity complex of inositol triphosphate with a phospholipase C pleckstrin homology domain. Celt 83 1037-1046, 1995. 

Pseudopterosin A is a member of a group of marine natural products which show potent antiinflammatory properties, but which are not prostaglandin biosynthesis inhibitors. Structurally similar to phosphatidyl inositol, they may function as phospholipase inhibitors, and, as such, may be the forerunners of a new class of therapeutic agents. 

Hydrolytic enzymes phospholipases in snake venoms, endogenous... 

Phosphatidylcholine Apply phospholipase C solution as a band, dry, apply sample solution to enzyme band, stop reaction with hydrochloric acid vapor. sn-l,2-Digly-cerides are produced. [43]... 

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. 

Eicosanoids, so named because they are all derived from 20-carbon fatty acids, are ubiquitous breakdown products of phospholipids. In response to appropriate stimuli, cells activate the breakdown of selected phospholipids (Figure 25.27). Phospholipase Ag (Chapter 8) selectively cleaves fatty acids from the C-2 position of phospholipids. Often these are unsaturated fatty acids, among which is arachidonic acid. Arachidonic acid may also be released from phospholipids by the combined actions of phospholipase C (which yields diacyl-glycerols) and diacylglycerol lipase (which releases fatty acids). 

LY311727 is an indole acetic acid based selective inhibitor of human non-pancreatic secretory phospholipase A2 (hnpsPLA2) under development by Lilly as a potential treatment for sepsis. The synthesis of LY311727 involved a Nenitzescu indolization reaction as a key step. The Nenitzescu condensation of quinone 4 with the p-aminoacrylate 39 was carried out in CH3NO2 to provide the desired 5-hydroxylindole 40 in 83% yield. Protection of the 5-hydroxyl moiety in indole 40 was accomplished in H2O under phase transfer conditions in 80% yield. Lithium aluminum hydride mediated reduction of the ester functional group in 41 provided the alcohol 42 in 78% yield. 

FIGURE 2.7 Production of second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) through activation of the enzyme phospholipase C. This enzyme is activated by the a- subunit of Gq-protein and also by Py subunits of Gj-protein. IP3 stimulates the release of Ca2+ from intracellular stores while DAG is a potent activator of protein kinase C. 

Phospholipase C, 24 pK0, 125, 144-145 Polar metabolites, 165 Polymorphisms, 4 Polypharmacology, 190-192 Pooled variance, 228 Populations, 226-228, 232 Positive agonism, 49 Potency... 

The second type of material includes spores, which may or may not produce disease symptoms but which can germinate in the insect gut and give rise to vegetative bacterial cells which in turn may produce, and exoenzymes such as phospholipases (lecithinases) or hyaluronidase. The phospholipases may produce direct toxic symptoms owing to their action on nervous or other phospholipid-containing tissue. Hyaluronidase breaks down hyaluronic acid and produces effects on animal tissue which are morphologically similar to the breakdown of insect gut wall in the presence of microbial insecticide preparations. 

Heat-labile soluble toxin Exoenzymes (phospholipases, hyaluronidase) Vegetative bacterial cells... 

PH domains consist of about 120 amino acid residues. They do not interact with other proteins, but associate with specific polyphosphoinositides. Consequently, PH domains appear to be important for localizing target proteins to the plasma membrane. Examples of PH domain-containing proteins include phospholipase C andpl20/RasGAP (Fig. 1). 

Two AR subtypes, Ax and A3, couple through G to inhibit adenylate cyclase, while the other two subtypes, A2a and A2B, stimulate adenylate cyclase through Gs or G0if (for A2a). The A2BAR is also coupled to the activation of PLC through Gq. Furthermore, each of these receptors may couple through the (3,y subunits of the G proteins to other effector systems, including ion channels and phospholipases. Levels of intracellular... 

TXA2 is produced by activated platelets via the sequential conversion of arachidonic acid by phospholipase A2, cyclooxygenase-1 (COX-1), and thromboxane synthase. Similar to ADP, TXA2 acts as a... 

Pasteurella multocida toxin (PMT) is the major pathogenic factor responsible for atrophic rhinitis, a disease which is characterized by bone loss in the nose of pigs. PMT is a 145 kDa single-chain exotoxin, which activates Goq protein (but not Gan) and stimulates phospholipase C 3. In addition, G12/i3 proteins and subsequently Rho pathways are activated. 

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