Phospholipids, phospholipase hydrolysis

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. 

P NMR studies on locust lipophorin indicate that a large proportion of the PLs reside on the surface of lipophorin (Katagiri et al., 1985). In addition to this study, the susceptibility of lipophorin phospholipids to hydrolysis by phospholipase A2 is consistent with a surface localization of PLs (Katagiri et al., 1985 Kawooya et al., 1991). Although the amphi-pathic nature of PLs makes this a logical conclusion, it has to be pointed out that the use of hydrolytic enzymes to demonstrate the location of any component would be valid only if a rearrangement of the lipid components in lipophorin does not occur on the time scale of the experiment. Because this condition is never met, a cautious interpretation of such data is necessary. 

The hydrolysis of glycerophospholipids ester bonds are regulated by a group of enzymes namely phospholipases. The site of action of every phospholipase is specific for every enzyme and is of great importance for the biological role of the phospholipids. Phospholipase A2 for example stereospeciftcally catalyzes the hydrolysis of xn-2 acyl ester bond, and plays an important role in the lung inflammation pathogenesis Fig. (5) LU5]. 

Phospholipid (PL) fatty acid composition and stereospecific distribution of 25 genetically modified soybean lines having a wide range of compositions were determined by GC and phospholipase hydrolysis (Wang et al., 1997). PL class proportions were affected by changes in overall fatty acid composition. PL fatty acid composition was changed with oil fatty acid modification, especially for palmitate, stearate, and linolenate. 

FIGURE 2. Post-receptor events initiated by H2- and H,-receptor activation by histamine (HIS). Adenylate cyclase (AC) stimulation occurs directly via H2-receptor activation or possibly indirectly via a calmodulin-calmodulin binding protein complex. Membrane inositol phospholipid (IPL) hydrolysis to inositol trisphosphate (IP3) is triggered by phospholipase C (PC) activation following H,-receptor stimulation. 

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). 

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. 

Two possible pathways for the biosynthesis of 2-AG have been proposed (1) a phospholipase C (PLC) hydrolysis of membrane phospholipids followed by a second hydrolysis of the resulting 1,2-diacylglycerol by diacylglycerol lipase or (2) a phospholipase Ai (PLA,) activity that generates a lysophospholipid, which in turn is hydrolyzed to 2-AG by lysophospholipase C (Fig. 5) (Piomelli, 1998). Alternative pathways may also exist from either triacylglycerols by a neutral lipase activity or lysophosphatidic acid by a dephosphorylase. The fact that PLC and diacylglycerol lipase inhibitors inhibit 2-AG formation in cortical neurons supports the contention that 2-AG is, at least predominantly, biosynthesized by the PLC pathway (Stella, 1997). However, a mixed pathway may also be plausible. 

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). 

Like proteases, phospholipases (PLs) are enzymes that catalyze the hydrolysis of a covalent bond, in this particular case of phospholipids. For this reason, a similar approach for the design of FRET... 

Parvalbumins Phospholipase A2 High-affinity calcium buffers Hydrolysis of phospholipids with the release of free... 

The family of heterotrimeric G proteins is involved in transmembrane signaling in the nervous system, with certain exceptions. The exceptions are instances of synaptic transmission mediated via receptors that contain intrinsic enzymatic activity, such as tyrosine kinase or guanylyl cyclase, or via receptors that form ion channels (see Ch. 10). Heterotrimeric G proteins were first identified, named and characterized by Alfred Gilman, Martin Rodbell and others close to 20 years ago. They consist of three distinct subunits, a, (3 and y. These proteins couple the activation of diverse types of plasmalemma receptor to a variety of intracellular processes. In fact, most types of neurotransmitter and peptide hormone receptor, as well as many cytokine and chemokine receptors, fall into a superfamily of structurally related molecules, termed G-protein-coupled receptors. These receptors are named for the role of G proteins in mediating the varied biological effects of the receptors (see Ch. 10). Consequently, numerous effector proteins are influenced by these heterotrimeric G proteins ion channels adenylyl cyclase phosphodiesterase (PDE) phosphoinositide-specific phospholipase C (PI-PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phospholipase A2 (PLA2), which catalyzes the hydrolysis of membrane phospholipids to yield arachidonic acid. In addition, these G proteins have been implicated in... 

Fig. 3.14. Mechanism of the formation of a noncovalently bound tetrahedral intermediate in the hydrolysis of phospholipids by phospholipase A2 [86]... 

Methods used to demonstrate the existence of membrane phospholipid asymmetry, such as chemical labelling and susceptibility to hydrolysis or modification by phospholipases and other enzymes, are rmsuitable for dynamic studies because the rates of chemical and biochemical reactions are of a different order compared to the transmembrane translocahon of the phospholipids. Indirect methods have therefore been developed to measure the translocation rate which are consequent on the loss of membrane phospholipid asymmetry. Thus time scales appropriate to rates of lipid scrambling under resting conditions or when the forces preserving the asymmetric phospholipid distribution are disturbed can be monitored. Generally the methods rely on detecting the appearance of phosphatidylserine on the surface of cells. Methods of demonstrating Upid translocation in mammalian cells has been the subject of a recent review (Bevers etal., 1999). 

Figure 4.12 Hydrolysis of a phospholipid (lecithin) in the lumen by a phospholipase. Lysolecithin is a lysophospholipid and is a detergent. At high concentrations it can damage membranes. It is also produced during repair of damaged phospholipids (Chapter 11)...

They act as local messengers in tissues and they provide communication between one cell type and another within a single tissue or an organ (i.e. they have a paracrine effect). They are synthesised from the polyunsaturated fatty acids that contain 20 carbons (e.g. arachidonate, eicosapentaenoic acids). These fatty acids are generated from the hydrolysis of membrane phospholipids by the activity of a phospholipase which releases the fatty acids from position 2 of the phospholipids. Their roles are described in detail below. 

The phosphorylated phospholipid, phosphatidylinositol bisphosphate, is present in cell membranes. On hydrolysis by a phospholipase, it produces two products, inositol trisphosphate and diacylglycerol (Figure 11.25), as follows ... 

Figure 11.30 Mechanisms of regulation of phospholipase A2. In all these processes described above, it is phospholipase A that carries out the hydrolysis of membrane phospholipid. Cytokines are local hormones produced by immune cells, T-lymphocytes and macrophages (Chapter 17). Other factors relate to shear stress in endothelial cells and those that stimulate release of granules from mast cells. Eicosanoids are present in the granules and they must be re-synthesised after degranulation in the mast cells. Here the enzymes described above must be present in mast cells.

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