Phospholipase substrate preference

A considerable amount of data has been reported on the substrate preference of the phospholipase C present in the organism Bacillus cereus. Interestingly, three phospholipases C have been isolated and purified, the first of which has high specificity for phosphatidylcholine, the second for phospha-tidylinositol, and the third for sphingomyelin (often termed sphingomyelinase). Similar substrate requirements have been noted in the phospholipase C isolated from other bacteria. 

Arisz, S.A., Valianpour, E, Van Gennip, A.H. and Munnik, T., 2003, Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation. Plant J. 34 595-604. 

FRET probes have not only been generated to measure the phospholipase activity but to study its substrate specificity as well. Several substrates of PLA2 with a variety of head groups and labeled with a BODIPY dye and a Dabcyl quencher were created by Rose et al. and tested against different PLAs in cells to determine substrate specificity and intracellular localization [137], The specificity of PLA2 isoforms towards the number of double bonds in the sn2 position was evaluated with a small series of PENN derivatives. It was demonstrated that the cytosolic type V PLA2 preferred substrates with a single double bond [138],... 

Belkner et al. [32] demonstrated that 15-LOX oxidized preferably LDL cholesterol esters. Even in the presence of free linoleic acid, cholesteryl linoleate continued to be a major LOX substrate. It was also found that the depletion of LDL from a-tocopherol has not prevented the LDL oxidation. This is of a special interest in connection with the role of a-tocopherol in LDL oxidation. As the majority of cholesteryl esters is normally buried in the core of a lipoprotein particle and cannot be directly oxidized by LOX, it has been suggested that LDL oxidation might be initiated by a-tocopheryl radical formed during the oxidation of a-tocopherol [33,34]. Correspondingly, it was concluded that the oxidation of LDL by soybean and recombinant human 15-LOXs may occur by two pathways (a) LDL-free fatty acids are oxidized enzymatically with the formation of a-tocopheryl radical, and (b) the a-tocopheryl-mediated oxidation of cholesteryl esters occurs via a nonenzymatic way. Pro and con proofs related to the prooxidant role of a-tocopherol were considered in Chapter 25 in connection with the study of nonenzymatic lipid oxidation and in Chapter 29 dedicated to antioxidants. It should be stressed that comparison of the possible effects of a-tocopherol and nitric oxide on LDL oxidation does not support importance of a-tocopherol prooxidant activity. It should be mentioned that the above data describing the activity of cholesteryl esters in LDL oxidation are in contradiction with some earlier results. Thus in 1988, Sparrow et al. [35] suggested that the 15-LOX-catalyzed oxidation of LDL is accelerated in the presence of phospholipase A2, i.e., the hydrolysis of cholesterol esters is an important step in LDL oxidation. 

Cation Requirements. While some of the phospholipases C found in bacteria appear to prefer Ca2+, there are many many reports supporting Zn2+ as the divalent cation of choice. There is some support for the fact that this enzyme is probably a metallo (Zn2+) protein which also requires Ca2+ for catalytic activity, but there is more evidence for the enzyme s ability to influence the surface charge on the micellar substrate system. 

Crude extracts of the ascidian Didemnum candidum exhibit strong inhibitory effects against phospholipase A2 in vitro. A search for the active component led to the discovery of ascidiatrienolide A 33 (20), an eicosatetraene derivative that is closely related to the didemnilactones 34-36 derived from the tunicate Didemnum moseley (27). The latter are endowed with high affinity to the leukotriene B4 receptor of human polymorphonuclear leucocyte membrane fractions. These fatty acid derivatives of marine origin provided yet another opportunity to validate our strategy for ( ,Z)-control outlined in the previous chapter based upon proper matching of the reactivity of the metathesis catalyst with the conformational preferences of a constrained substrate (22). 

McLean et al. (110) have examined the role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids. Results showed that the increase in rate of hydrolysis of peroxidized phospholipid substrates catalyzed by phospholipase A2 is largely because of a preference for peroxidized phospholipid molecules as substrates, and that peroxidation of the host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids. 

Phospholipase D catalyzes the hydrolysis of phospholipids to produce phosphatidic acid and the corresponding polar head group [215]. In most systems, including neutrophils, phosphotidylcholine is the preferred substrate. Two mammalian isoforms of the enzyme have been cloned, but PLD purified from human neutrophils displays different biochemical characteristics, suggesting it represents a unique isoform [139, 215], In the presence of primary alcohols, PLD catalyzes a transphosphatidylation reaction that produces the corresponding phos-phatidylalcohol. This transphosphatidylation reaction effectively competes with hydrolysis, and thus alcohols such as ethanol and 1-butanol are frequently used experimentally as inhibitors of PLD-catalysed PA production. 

Phospholipase A enzymes - particularly those from snake venoms or digestive secretions - have been widely studied. Phospholipase Ai is found in microsomal and liposomal fractions (cf. Newkirk and Waite, 1971 Gatt, 1968). It specifically deacylates phosphatidylcholine or phosphatidylethanolamine at the 1-position. Both these substrates are hydrolysed at the same rate by the adrenal medulla lysosomal enzyme, but that from brain prefers phosphatidylcholine as substrate. Detergents will increase phosphatidylethanolamine hydrolysis by the brain enzyme. A phospholipase Ai (which is relatively specific for phosphatidylglycerol) has been reported from the spores of some bacteria (Raybin et aL, 1972), but most bacterial enzymes are unspecific for either the 1- or the 2-positions. 

Figure 1. Structure of phosphatidylinositol. Phosphatidylinositol (Ptdins) constitutes about 10% of the total phospholipids in eukaryotic cells and is the precursor of the other phosphoinositides (polyphosphoinositides) through sequential phosphorylations by specific kinases. As indicated, its inositol head group can be phosphorylated at three positions (D-3, D-4 and D-5) by specific kinases in vivo. The cleavage by phosphoinositide-specific phospholipase C (PLC), which has as its preferred substrate PtdIns(4,5)P2, is also shown. PI3K, phosphoinositide 3-kinase. PI-K II and III, phosphatidylinositol kinase types II and III. PIP-K I, phosphatidylinositol monophosphate kinase type I. PIP-K II, phosphatidylinositol monophosphate kinase type II.

Eicosapentaenoic add (Table 6) is easily incorporated into the cell membrane-bound phospholipids. It is a poor substrate for the cyclooxygenase/hydroper-oxidase enzymatic system, and only very small amounts of endproducts with three double bonds will be formed when compared with the relative effectiveness of arachidonate metabolism. In contrast, dcosapentaenoic add is a preferred substrate for product generation by the S-lipoxygenase in subcellular fractions of human and animal neutrophils [66,87], whereas docosahexaenoic add is also a markedly inferior substrate for leukotriene synthesis. Eicosapentaenoic acid appears to inhibit phospholipase activity, resulting in a decrease of aradiidonic acid release ([67] Fig. 4). Moreover, simultaneously with thromboxane A3 and prostaglandin I3 generation from eicosapentaenoic add, the synthesis of thromboxane A2 and prostacyclin (PGIj) from aradiidonic acid [47] decreases, mainly due to inhibitory effects on PGH synthase activity (Fig. 4). 

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