Phospholipid substrates

MICELLAR SUBSTRATES. Phospholipids in micelles are frequently found to be more active substrates in lipolysis than those phospholipids residing in a lipid bilayer". Dennis first described the use of Triton X-100 to manipulate the amount of phospholipid per unit surface area of a micelle in a systematic analysis of the interfacial interactions of lipases with lipid micelles. Verger and Jain et al have presented cogent accounts of the kinetics of interfacial catalysis by phospholipases. The complexity of the problem is illustrated in the diagram shown in Fig. 2 showing how the enzyme in the aqueous phase can bind to the interface (designated by the asterisk) and then become activated. Once this is achieved, E catalyzes conversion of S to release P. ... 

Phospholipases (PLs) are a ubiquitous group of enzymes that share the property of hydrolyzing a common substrate, phospholipid. Nearly all share another property they are more active on aggregated substrate above the phospholipid s critical micellar concentration (cmc). As shown in Fig. 1, phospholipases have very low activity on monomeric substrate but become activated when the substrate concentration exceeds the cmc. The properties of phospholipids that define the aggregation state (micelle, bilayer vesicle, hexagonal array, etc.) are described in Chapter 1. 

Remodeling of the fatty acid composition of the sn- position of phosphatidylcholine or A-acyltransferase substrate phospholipids will result in a changed composition of the NAEs. This is seen in severe neuronal injury, when the A-stearoylethanolamine accumulates to a greater extent than the other NAEs (55,56). 

As mentioned above, phospholipases are distinguished from general esterases by the fact that they interact with interfaces in order to function. The difference in reaction velocity with substrate concentration for these two types of enzymes is illustrated in Figure 7.6. Whereas esterases show classical Michaelis-Menton kinetics, the phospholipases show a sudden increase in activity as the substrate (phospholipid) concentration reaches the critical micellar concentration (CMC) and the molecules tend to form aggregates or micelles with the polar ends in the aqueous environment... 

Mammals synthesize phosphatidylserine (PS) in a calcium ion-dependent reaction involving aminoalcohol exchange (Figure 25.21). The enzyme catalyzing this reaction is associated with the endoplasmic reticulum and will accept phosphatidylethanolamine (PE) and other phospholipid substrates. A mitochondrial PS decarboxylase can subsequently convert PS to PE. No other pathway converting serine to ethanolamine has been found. 

Tyrosine phosphorylated IRS interacts with and activates PI 3-kinase [3]. Binding takes place via the SRC homology 2 (SH2) domain of the PI 3-kinase regulatory subunit. The resulting complex consisting of INSR, IRS, and PI 3-kinase facilitates interaction of the activated PI 3-kinase catalytic subunit with the phospholipid substrates in the plasma membrane. Generation of PI 3-phosphates in the plasma membrane reemits phospholipid dependent kinases (PDKl and PDK2) which subsequently phosphorylate and activate the serine/threonine kinase Akt (synonym protein... 

Lipid phosphate phosphohydrolases (LPPs), formerly called type 2 phosphatidate phosphohydrolases (PAP-2), catalyse the dephosphorylation of bioactive phospholipids (phosphatidic acid, ceramide-1-phosphate) and lysophospholipids (lysophosphatidic acid, sphingosine-1-phosphate). The substrate selectivity of individual LPPs is broad in contrast to the related sphingosine-1-phosphate phosphatase. LPPs are characterized by a lack of requirement for Mg2+ and insensitivity to N-ethylmaleimide. Three subtypes (LPP-1, LPP-2, LPP-3) have been identified in mammals. These enzymes have six putative transmembrane domains and three highly conserved domains that are characteristic of a phosphatase superfamily. Whether LPPs cleave extracellular mediators or rather have an influence on intracellular lipid phosphate concentrations is still a matter of debate. 

Phosphatidylinositol (abbreviated Ptdlns, or PI) is a minor class of phospholipids composed of glycerol, fatty acids and inositol. Pis are found in the cytosolic side of eukaryotic cell membranes. They are substrates fora large number of enzymes which are involved in cell signalling. 

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.

Dihydroxyacetone phosphate (82) is a substrate for a-glycero-phosphate dehydrogenase, aldolase, and triose phosphate isomerase, and its O-alkyl ethers are intermediates in the biosynthesis of phospholipids. In neutral aqueous solution at 20 °C, dihydroxyacetone phosphate exists as an equilibrium mixture of the keto (82), gem-d o (83), and enol (84) forms, as shown by n.m.r. spectroscopy. The proportion of (82) to (83)... 

It is possible that nematode-secreted AChEs act on alternative substrates to ACh. We had previously suggested, on the basis of structural similarity, that platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, might represent such an alternative substrate (Blackburn and Selkirk, 1992b) but subsequent studies demonstrated that purified AChEs did not cleave PAF, and the enzyme responsible for this activity in secreted products of N. brasiliensis, PAF acetylhydrolase, was purified and defined as a distinct heterodimeric protein (Grigg et al., 1996). Although an open mind on the subject sould be kept, the strict substrate specificity of the nematode-secreted AChEs suggests that they most likely act on ACh alone. 

Fig. 10.7 RNA synthesis in vesicles. Membrane permeability can be regulated by choosing the correct chain length of the fatty acids in the phospholipids. Short chains (a) make the bilayer so unstable that even large molecules such as proteases can enter the vesicle interior and damage the polymerase. Carbon chains which are too long (b) prevent the entry of substrate molecules such as ADR RNA polymerisation in the vesicle occurs only with C14 fatty acids (c)...

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

This encapsulation procedure gives the highest activity for the lipases. The lecithin/ amines mixture structuring the pore network leads to a suitable phospholipids bilayer-like environment, which avoids the necessity to create an interface by substrate assembly. Monduzzi and coworkers compared the activity of lipase that was immobilized on SBA-15 physically, or chemically with glutardialdehyde [200]. 

Proteins from cDNA libraries are used for measurement of kinase substrate specificity and identification of phospholipid-binding proteins. 

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