Phospholipase characteristics

Phospholipase D is widely distributed in bacteria, fungi, plants and animals, and is present in almost all mammalian cells [3]. In mammals, it occurs as alternatively spliced products of two genes (PLD1 andPLD2) (Fig. 3). Most mammalian cells express different levels of both isoforms. Both PLD1 and PLD2 have four conserved sequences (I-IV), and sequences I and IV contain the HXKX4D (HKD) motif that is characteristic of the PLD superfamily, which includes bacterial endonucleases, phospholipid synthases, viral envelope... 

The characteristics of the four major classes of histamine receptors are summarized. Question marks indicate suggestions from the literature that have not been confirmed. AA, arachidonic acid DAG, diacylglycerol Iko,2+, calcium-activated potassium current IP3, inositol 1,4,5-trisphosphate NHE, sodium-proton exchange, PKC, protein kinase C NO, nitric oxide PTPLC, phosphoinositide-specific phospholipase C TXA2, thromboxane A2. Has brain-penetrating characteristics after systemic administration. 

Phospholipase D Cabbage Triton X-lOO/phosphotidyl choline/diethyl ether Enzyme characteristics [87]... 

Natural fats and oils can be used directly in products, either individually or as mixtures. In many cases, however, it is necessary to modify their properties, particularly their melting characteristics, to make them suitable for particular applications. Therefore, the oils and fats industry has developed several modification processes using enzyme technology. In particular, lipases (and lately cutinases), phospholipases and pectinases can be used for interesterification processes, ester syntheses and in olive-oil extraction. 

Phosphohpases of type Cy are activated by receptor tyrosine kinases (see Chapter 8), and thus phosphohpase Cy is involved in growth factor controlled signal transduction pathways. The receptor tyrosine kinases (see Chapter 8) phosphorylate the enzyme at specific tyrosine residues and initiate activation of the enzyme. Characteristic for the structure of phospholipase Cy is the occurrence of SH2 and SH3 domains (see Chapter 8). These represent protein modules that serve to attach further partner proteins. 

T FIGURE 10-18 Arachidonic acid and some eicosanoid derivatives. (a) In response to hormonal signals, phospholipase A2 cleaves arachidonic acid-containing membrane phospholipids to release arachidonic acid (arachidonate at pH 7), the precursor to various eicosanoids. (b) These compounds include prostaglandins such as PCE, in which C-8 and C-12 of arachidonate are joined to form the characteristic five-membered ring. In thromboxane A2/ the C-8 and... 

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidylcholine) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure. 

Prostaglandins have been implicated both in the induction of inflammation and in its relief. In inflammation small blood vessels become dilated, and fluid and proteins leak into the interstitial spaces to produce the characteristic swelling (edema). Many polymorphonuclear leukocytes attracted by chemotactic factors that include LTB4309 (Chapter 19) migrate into the inflamed area, engulfing dead tissue and bacteria. In this process lysosomes of the leukocytes release phospholipase A, which hydrolyzes phospholipids and initiates the arachidonate cascade. The leukotrienes that are formed promote the inflammatory response. However, cAMP can suppress inflammation, and PGE2 has a similar effect. Indeed, E prostaglandins, when inhaled in small amounts, relieve asthma. 

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. 

Lamura, E., et al. 1997. Compartmentalisation and characteristics of a Ca2+-dependent phospholipase A2 in human colon mucosa. Biochem Pharmacol 53 1323. 

Simpson LL (2000) Identification of the characteristics that underlie botulinum toxin potency implications for designing novel drugs. Biochimie 82 943-53 Simpson LL, Lautenslager GT, Kaiser II, Middlebrook JL (1993) Identification of the site at which phospholipase A2 neurotoxins localize to produce their neuromuscular blocking effects. Toxicon 31 13-26... 

Relevant quality control should not be restricted to the usual triad of activities of pancreas lipase, a-amylase, and trypsin, but should be extended to the content of colipasc, the activities of the two other lipolytic enzymes present in pancreatine (phospholipase Aj and carboxylester lipase), and the dissolution characteristics of enteric-coated preparations as a function of time and pH (Fig. 16). The availability of such information will certainly contribute to a better tailoring of flic management of maldigestion in the individual patient and to a more appropriate correction of the obligate nonphysio logical route of delivery of these enzyme supplements. 

A very satisfactory source of phospholipase A2 is the venom of the snake, Crotalus adamanteus (Eastern diamondback rattlesnake). This venom can be obtained in lyophilized form from commercial suppliers such as Miami Ser-pentarium (Miami, FL). Of importance, the lyophilization process does not alter the chemical, physical, or enzymatic characteristics of the original venom obtained from this snake. 

Figure 4. Linear representation of the Arabidopsis PIPKs and PLCs. The Arabidopsis PtdlnsP 5-kinases are most similar to the human type I PtdlnsP 5-kinases. There are 11 putative type I /I/PtdlnsP 5-kinases in Arabidopsis arranged in two subfamilies based on size. Subfamily B contains X/PIPK1-9, all of which contain membrane occupation and recognition nexus (MORN) repeats. /1/PIPK10-11 are in Subfamily A with molecular weights less than that of the members of subfamily B and contain no MORN repeats. The Arabidopsis phosphoinositide specific phospholipase C family is most similar to the animal PLC . There are seven functional PI-PLCs in Arabidopisis (Hunt et al., 2004). All isoforms contain EF-hand motifs, the X and Y catalytic domains characteristic of PI-PLCs and a C2 lipid-binding domain.

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