Phospholipases

Phospholipases are potentially important in milk and milk products because of their ability to degrade the phospholipids of the milk fat globule membrane, thereby increasing the susceptibility of the milk fat to lipolytic attack (Fox et al., 1976 Griffiths 1983). 

Cows milk LPL has phospholipase Ai activity (Scow and Egelrud, 1976), but its action on milk phospholipids has not been recorded. Freshly secreted goats milk has been shown to have phospholipase A activity (Long and Patton, 1978) but it is not known whether this can be attributed to the LPL of that milk. Human milk contains an acid sphingomylinase C, as well as ceramidase activity provided by the bile salt-stimulated lipase present (Nyberg et al., 1998). 

Several psychrotrophic bacteria produce extracellular phospholipases, the most prevalent in milk being pseudomonads (particularly P. fluorescens), Alcaligenes, Acinetobacter, and Bacillus species (Fox et al., 1976 Owens, 1978a Phillips et al., 1981). Most of these produce phospholipase C, some produce phopholipase Ai and some produce both types (Deeth, 1983). Ser-ratia spp. have been shown to produce only phospholipase A (Deeth, 1983), while P. fragi has been reported not to produce phospholipases (Kwan and Skura, 1985). Phospholipase C from some pseudomonads has been purified and characterised (Doi and Nojima, 1971 Sonoki and Ikezawa, 1975 Stepa-niaketa/., 1987a Ivanov etal., 1996). Like the lipases, many of these enzymes have considerable heat stability and are not destroyed by pasteurization 

These enzymes are denoted as phospholipases, lysophospholipases or glycohpid hydrolases, depending on the substrate. 

Phospholipase C. It hydrolyzes lecithin to a 1,2-diacylglyceride and phosphoryl chohne. The enzyme is found in sndkQ venom and in bacteria. 

Phospholipase D. This enzyme cleaves the chohne group in the presence of water or an alcohol, such as methanol, ethanol or glycerol, yielding free or esterified phosphatidic acid. For example  

Phospholipase A. The enzyme is present together with phosphohpase A2 in many mammals and bacteria. It cleaves specifically the sn-1 ester bonds of diacylphosphatides (Formula 3.53). 

Phospholipase A2. Enzymes with sn-2 specificity isolated form snake and bee venoms. They are very stable, are activated by Ca +-ions and are amongst the smallest enzyme molecules (molecular weight about 14,000). 

In platelets, PAF triggers multiple signaling cascades (Fig. 3). Thus, activation of phospholipases, protein kinases, Ca mobilization, MAP kinases mediated pathways have 

Initially it was demonstrated in rabbit platelets that PAF activated turnover of phosphoinositides monitored by labeling of PI, PIP and PIPj ( Shukla and Hanahan, 

Subsequently, it was shown using [%] inositol labeled rabbit platelet that PAF activated phospholipase C pathway resulting in the generation of [ ] inositol phoshates 

PAF receptor is coupled to the activation of both protein kinase C and tyrosine kinase (Shukla, 1992, Izumi and Shimizu, 1995). PAF receptor antagonists block both pathways. The activation of PKC is through the Imown PLC generated diglyceride -t- Ca pathway (leyasu et. al. 1982). Activation of PKC in platelets (eg. by phrobol myristate acetates) inhibited PAF stimulation of platelets. On the other hand PKC inhibitors (eg. staurosporine) enhanced PAF stimulated IP, production. In rabbit platelets PKC activation by PAF was suggested to be independent of enzyme translocation (Pelech et. al. 1990). Human platelets have 6 PKC isozymes (alpha, p, 8, zeta, eta and theta). PAF stimulated 200% and 175% increase in the levels of membrane - bound PKC eta and theta 

Role of endogenously activated type 1, protein phosphatese 1 (PPl) and type 2A (PP2A) in the signal transduction pathway of PAF stimulated rabbit platelets has been studied. Calyculin A, an inhibitor of PPl and PP2A, caused inhibition of Ca influx by PAF and correlated with inhibition of Ins (1,4,5) P, formation (Muiphy and Westwick, 1994). It is therefore abundantly clear that both PKC and tyrosine kina% are integral components of PAF receptor signaling pathways. PAF is one of the first G-protein coupled receptors reported to be coupled to tyrosine kinase (Dhar et.al. 1990). 

Based mainly on cell-free assays, 10 enzymatic activities that degrade phospholipids, intermediates in the phospholipid biosynthetic pathway, or triacylglycerol have been reported (Table 2). The detergent-resistant phospholipase A, (encoded by pldA) of the outer membrane, characterized by Nojima and colleagues (Y. Nakagawa, 1991), is the most studied of these enzymes. This enzyme is unusually resistant to inactivation by heat and ionic detergents 

Phospholipase A1 pldA Outer membrane Phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and lyso derivatives 

Phosphatidic acid phosphatase Membrane Phosphatidic acid 

1- acyl isomer. This lysophospholipase also catalyzes the transfer of fatty acids from 

2- acyl-glycerophosphoethanolamine to PtdGro to form acyl-PtdGro. 

This chapter will focus on the families of enzymes that oxidize PUFA, their cellular location, function and substrate specificity, and introduce the role they and their enzymatic products play in cellular health and pathophysiological conditions. 

LysoPLA2 has low molecular weight (45 kDA) and exhibits Ca + independent PLA2, transacylase and 1-o-acylceramide synthase activities, with specificity for PC- and PE-esterified unsaturated fatty acids (Abe and Shayman 1998 Dennis et al. 

A PLD with specificity for N-arachidonyl phosphatidylethanolamines is involved with production of the endocannabinoid anandamide (AEA), and other PUFA-ethanolamides (Fonseca et al. 2013). 

Interestingly, the activity of many phospholipases can be affected by reactive oxygen species (ROS), thus impacting upon the related signaling pathways (Korbecki et al. 2013). Ultraviolet radiation (UVR)-induced ROS formation has been shown to increase the activity and expression of CPLA2 with concomitant stimulation of the A A cascade in skin, while the activity of NADPH oxidases are affected by A A, DAG, and PA, produced by PLA2, PLC, and PLD, respectively (Chen et al. 1996 Cummings et al. 2002 Frey et al. 2002 Pendyala et al. 2009). 

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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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