Phospholipase enzymes

Hydrolytic enzymes phospholipases in snake venoms, endogenous... 

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. 

Figure 3.1 Schematic representation of a generic excitatory synapse in the brain. The presynaptic terminal releases the transmitter glutamate by fusion of transmitter vesicles with the nerve terminal membrane. Glutamate diffuses rapidly across the synaptic cleft to bind to and activate AMPA and NMDA receptors. In addition, glutamate may bind to metabotropic G-protein-coupled glutamate receptors located perisynaptically to cause initiation of intracellular signalling via the G-protein, Gq, to activate the enzyme phospholipase and hence produce inositol triphosphate (IP3) which can release Ca from intracellular calcium stores...

Fig. 4.12(a). An outline structure of a protein (here the enzyme phospholipase A2), showing a-helical runs of amino acids as cylinders (A-E) and anti-parallel P-sheet runs as heavy black arrows. Disulfide cross-links are shown (the enzyme is extracellular), and runs of no a/p secondary structure appear as thin lines. The structure is relatively immobile, and binds calcium in a constrained loop. (Reproduced with permission from Professor J. Drenth.)... 

A colorimetric assay for lecithin and choline was described by Kotsira and Klonis (1998) using two enzymes (phospholipase and choline oxidase) and an indicator dye conjugate (bromothymol blue-glutathione) co-immobilised on a glutaraldehyde-activated polyacrylamide transparent gel. The change of the... 

B. Waszkowycz, I. H. Hiller, N. Gensmantel, D. W. Payling, A Combined Quantum Mechanical/Molecular Mechanical Model of the Potential Energy Surface of Ester Hydrolysis by the Enzyme Phospholipase A2 ,. /. Chem. Soc., Perkin Trans. 2 1991, 225-231. 

Synthesizing Enzymes Phospholipase D Phospholipase C Diacylglycerol lipase... 

Anandamide is believed to be synthesized from a phospholipid precursor, /V-arachidonoyl-phosphatidylethanolamine, catalysed by phospholipase D (Di Marzo et al. 1998). The other proposed route of synthesis is from condensation of arachidonic acid and ethanolamine, although this has yet to be demonstrated in living cells. 2-AG is formed in a calcium-dependent manner, and mediated by the enzymes phospholipase C and diacylglycerol lipase (Kondo et al. 1998 Stella et al. 1997). 

G proteins are divided into several types, depending on their effects. Stimulatory G proteins (Cs) are widespread. They activate adenylate cyclases (see below) or influence ion channels. Inhibitory G proteins (Cj) inhibit adenylate cyclase. G proteins in the Gq family activate another effector enzyme—phospholipase c (see p. 386). 

The eicosanoids, so called because of their derivation from a 20-carbon unsaturated fatty acid, arachidonic acid (eicosatetraenoic acid), are obtained from membrane phospholipids and synthesized de novo at the time of cellular stimulation. Arachidonic acid is cleaved from membrane-bound phosphatidylcholine by the enzyme phospholipase A2. Alternatively, arachidonic acid may be derived by the sequential actions of phospholipase C and diacylglyceryl lipase. Arachidonic acid can then follow either of two enzymatic pathways that result in the production of inflammatory mediators. The pathway initiated by cyclooxygenase (COX) produces prostaglandins the lipoxygenase pathway generates leukotrienes (Fig. 36.2). 

Drugs, particularly organic bases, may release histamine from mast cells by physically displacing the amine from its storage sites. Morphine, codeine, d-tubocu-rarine, guanethidine, and radiocontrast media can release histamine from mast cells. Basic polypeptides, such as bradykinin, neurotensin, substance P, somatostatin, polymyxin B, and the anaphylatoxins resulting from complement activation, also stimulate histamine release. Venoms often contain basic polypeptides as well as the histamine-releasing enzyme phospholipase A. 

They also influence the inflammatory response by reducing the prostaglandin and leukotriene synthesis that results from activation of enzyme phospholipase A. ... 

Eurther work on the isolation of related compounds from mammalian sources, which spanned several decades, led to the identification of a large group of structurally related substances. Investigations on their biosynthesis made it evident that aU eventually arise from the oxidation of the endogenous substance, arachidonic acid. The individual products induce a variety of very potent biological responses, with inflammation predominating. Arachidonic acid, once freed from lipids by the enzyme phospholipase A2, can enter one of two branches of the arachidonic acid... 

Lipid hydroperoxides can be removed by reaction with GSH catalyzed by GSH peroxidase. The enzyme phospholipase A2 has been proposed to have a role in the detoxication of phospholipid hydroperoxides by releasing fatty acids from peroxidized membranes. 

Stretch-activated proteins in animal cell membranes that are candidates for osmosensing activity include mechanosensitive ion channels and the membrane-localized enzyme phospholipase A2 (PLA2). The former proteins remain to be conclusively linked to osmosensing. Activity of PLA2 is sensitive to packing of the lipid bilayer of the cell and is responsive to osmotic changes, two attributes that mark it as a prime candidate for a stretch-activated sensor (Lehtonen and Kinnunen, 1995). 

Finally, though only a direct chemical hyrolysis was described above for liberation of choline, it is possible to employ the enzyme phospholipase D to release choline from intact phosphatidylcholine with the concomitant formation of phosphatidic acid. 

A most provocative observation by Bruzik et al. (1983) using a mixture of the diastereoisomers (RP)-DPPSC and (Sp)-DPPsC was the ability of the enzyme, phospholipase A2, to distinguish between the isomers. This was quite unexpected since the P atom, which is five bonds removed from the susceptible carboxyl function, should have little influence on the course of the reaction. In any event, this reaction sequence can be described in the following... 

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. 

Besides these effects of AlF, on G protein systems, aluminum has its own actions on the phosphoinositide signaling pathway. Aluminum specifically inhibits the Ca2+-dependent enzyme phospholipase C which acts on PIP2. It was found that aluminum chloride inhibited the hydrolysis of PIP2 in a concentration-dependent manner with an IC(50) slightly above 100 pM [56]. The inhibition observed is competitive in nature with the substrate PIP2 [57]. 

Scorpionid secretions represent a mixture of neurotoxic polypeptide toxins, proteolytic and hemolytic enzymes (phospholipases A, acetylcholinesterases, ribonucleases, hyaluronidases), and biogenic amines (serotonin, tryptamine, histamine). The polypeptide toxins (the so-called scorpamines) contain fewer than 40 or 60-76 mostly alkaline and aromatic amino acids stabilized by four disulfide bridges.20 96... 

The agonist activity of serotonergic compounds at the 5-HT2 receptor was determined in vitro using the ability of the compounds to stimulate the production of [3H]inositol phosphates in [3H]myo-inositol-labeled A7r5 rat vascular smooth muscle cells by their ability to activate the enzyme phospholipase C. 

EnzyMax A process for degumming vegetable oils by use of the enzyme phospholipase A2, which converts water-insoluble phospholipids into hydratable phospholipids that can be extracted with water. Developed by Lurgi and first installed at Shenzen, China. 

---