Phospholipase substrates

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. 

Among the substrates of Src are other nonreceptor PTKs (e.g., Fak, Syk, and Tec kinases), RTKs (e.g. EGF and PDGF receptors), phospholipase Cy, PI3-kinase, phosphatases (e.g., SHP-2 and PP2A), and adaptor (e.g., She and Cbl) as well as focal adhesion proteins (e.g., paxillin, pl30Cas andtensin). Src-mediated phosphorylation either modulates enzymatic activity of... 

There are three groups of eicosanoids that are synthesized from C20 eicosanoic acids derived from the essential fatty acids linoleate and a-linolenate, or directly from dietary arachidonate and eicosapentaenoate (Figure 23-5). Arachidonate, usually derived from the 2 position of phospholipids in the plasma membrane by the action of phospholipase Aj (Figure 24-6)—but also from the diet—is the substrate for the synthesis of the PG2, 1X2 series (prostanoids) by the cyclooxygenase pathway, or the LT4 and LX4 series by the lipoxygenase pathway, with the two pathways competing for the arachidonate substrate (Figure 23-5). 

Figure 24-6. Sites of the hydrolytic activity of phospholipases on a phospholipid substrate.

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],... 

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

Zaikova, T. (2001). Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug. Chem. 12, 307-313. 

Wichmann, O., Gelb, M. H. and Schultz, C. (2007). Probing Phospholipase A(2) with Fluorescent Phospholipid Substrates. Chembiochem 8, 1555-1569. 

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. 

In contrast to their more highly expressed counterparts, the 3-phosphoinositides do not serve as substrates for phospholipase C (PLC), the enzyme known to be activated in stimulated phosphoinositide turnover. This observation indicates that the 3-phosphoinositides themselves, rather than their breakdown products, are likely to... 

This can be illustrated by known interactions between the cAMP and Ca2+ pathways. A first messenger that initially activates the cAMP pathway would be expected to exert secondary effects on the Ca2+ pathway at many levels via phosphorylation by PKA. First, Ca2+ channels and the inositol trisphosphate (IP3) receptor will be phosphorylated by PKA to modulate intracellular concentrations of Ca2+. Second, phospholipase C (PLC) is a substrate for PKA, and its phosphorylation modulates intracellular calcium concentrations, via the generation of IP3) as well as the activity of PKC, via the generation of DAG, and several types of CAMK. Similarly, the Ca2+ pathway exerts potent effects on the cAMP pathway, for example, by activating or inhibiting the various forms of adenylyl cyclase expressed in mammalian tissues (see Ch. 21). 

Prior to being able to study the function and mechanism of an enzyme, it is essential that suitable assays be available to monitor enzyme activity toward different substrates and to determine the kinetic parameters kcat and Km for the reactions. A brief overview of the known assays for the evaluation of PLCB(. activity is thus appropriate. The ideal assay for a phospholipase C would utilize a phospholipid substrate, not an analogue with a modified headgroup or side chains. Such an assay should be sensitive to minimize the quantities of enzyme and substrates that would be required, and it should be convenient to implement so that analyses may be readily performed. 

Massing U, Eibl H (1994) Substrates for phospholipase C and sphingomyelinase from Bacillus cereus. In Woolley P, Petersen SB (eds) Lipases. Their structure, biochemistry and application. Cambridge University Press, Cambridge, p 225... 

The phospholipases (PLC) isozymes cleave the phosphodiester bond in phos-phatidyl-inositol-4,5-bisphosphate (PIP2) releasing two second messenger molecules inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) as shown before. The /1-isozyme are controlled by the Ga or G y subunits of the heterotrimeric G-proteins coupled to surface receptors. The y-isozymes are substrates for tyrosine kinases, such as growth factors. 

The role of protein kinase C in many neutrophil functions is undisputed and has been recognised for some time. For many years it was believed that the source of DAG, the activator of protein kinase C, was derived from the activity of PLC on membrane phosphatidylinositol lipids. Whilst this enzyme undoubtedly does generate some DAG (which may then activate protein kinase C), there are many reasons to indicate that this enzyme activity is insufficient to account for all the DAG generated by activated neutrophils. More recently, experimental evidence has been provided to show that a third phospholipase (PLD) is involved in neutrophil activation, and that this enzyme is probably responsible for the majority of DAG that is formed during cell stimulation. The most important substrate for PLD is phosphatidylcholine, the major phospholipid found in neutrophil plasma membranes, which accounts for over 40% of the phospholipid pool. The sn-1 position of phosphatidylcholine is either acyl linked or alkyl linked, whereas the sn-2 position is invariably acyl linked. In neutrophils, alkyl-phosphatidylcholine (1-0-alky 1-PC) represents about 40% of the phosphatidylcholine pool (and is also the substrate utilised for PAF formation), whereas the remainder is diacyl-phosphatidylcholine. Both of these types of phosphatidylcholine are substrates for PLD and PLA2. 

Figure 6.19. Products of phosphatidylcholine metabolism. Phosphatidylcholine is metabolised to phosphatidic acid via the activity of phospholipase D. The phosphatidic acid generated in this way may then be converted into diacylglycerol via phosphatidate phospho-hydrolase (which is inhibited by propranolol), and the enzyme diacylglycerol kinase may regenerate the phosphatidic acid. Phospholipase D may also catalyse the transphosphati-dylation of primary alcohols, such as ethanol and butanol, at the expense of the natural substrate, phosphatidylcholine. Thus, primary alcohols can prevent phosphatidic acid production via this route.

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