1-Acyl-lyso

Our laboratory has been studying the kinetics (1) and mechanism of action (2) of phospholipase A2 which catalyzes the hydrolysis of the fatty acyl chain in the sn-2 position of phospholipids to give the 1-acyl lyso-phospholipid product ... 

Phospholipase AjS (PLAjs) hydrolyze the sn-1 fatty acid residue from phospholipids and produce mostly saturated or mono-unsaturated fatty acids and 2-acyl-lyso-phosphohpids. PLAjS have not attracted as much attention as mammalian acylhy-drolases, PLA2S, which are involved in the production of bio-active hpids such as prostanoids and platelet-activating factor (PAP) [1, 2]. 

Fig. Z5 A model of production of 2-acyl-lyso-PA by mPA-PLAiS and consequent activation of LPAj/EDG/ by 2-acyl-lysoPA. mPA-PLAla and -p hydrolyze PA generated as a result of...

However, a recent study (Sun et al. 2004) also highlights another possible way for NAPEs to be transformed into NAEs, at least in cell-free homogenates, i.e. via the sequential action of a group IB secretory phospholipase A2 (PLA2), with the formation of N-acyl-l-acyl-lyso-PE, followed by the action of a lyso-PLD enzyme distinct from the known NAPE-PLD (Fig. 2). 

Each MGDG molecular species was hydrolyzed by purified lipase A1 from Rhizopus arrhizus (Boehringer) according to [8]. The products obtained, 2-acyl lyso MGDG and free fatty acids liberated fiom position 1, were separated by TLC. 

Hydrolysis that occurs to some degree on autoclaving and storage results in the production of corresponding lyso-compounds, phospholipids without one of the acyl chains. These materials are called lyso- because, on their own, lyso-compounds produce lysis of red blood cells and are generally regarded as toxic. Analytically it is not difficult to demonstrate that there are significant quantities of these compounds present in injectable emulsions but there have never been any clinical reports of toxicity. The probability is that these materials form some type of association complex with the other phospholipids present and are not available on their own to exert any effect which would result in toxicity. 

Glycerophospholipids contain a glycerol skeleton to which two fatty acids are esterified saturated fatty acids occupy mostly sn-position 1, whereas unsaturated fatty acids are mainly present on sn-position 2. The third hydroxyl is linked to a phosphate group to which an organic base is mostly esterified (Fig. 1). The most important components of soybean lecithin are phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Phosphatidic acid (PA) may become important due to the presence of phospholipase D this enzyme slowly converts PC into PA in vegetable lecithins. Phosphatidylserine (PS), phosphatidylglycerol (PG), and lyso-phosphatidylcholine (LPC) are known as minor components lysophospholipids contain only one acyl group per molecule. Besides, ether phospholipids occur in which one or both fatty acyl... 

Hydrolysis and oxidation are the two primary degradation routes to which liposomal phospholipids are susceptible. Hydrolytic attack at the fatty acyl carbonyl will produce free fatty acids and lysophosphatides [e.g., lyso-phosphatidyl choline (LPC)]. Hydrolysis generally follows Lrst-order... 

Reiter et al. (43) have shown that a second enzyme can also act to degrade SGG. They found that secondary lysosomes from rat liver contained not only arylsulfatase A, but also a lipase activity that could act to de-acylate SGG. Under the conditions used, more product was formed by the action of the lipase on SGG than by the action of arylsulfatase A. These workers also found that the latter enzyme could use the lyso-SGG as a substrate. It would be... 

A2 in mixed phospholipid systems and the kinetics and migration of the acyl and phosphorus group of the lyso-phospholipid products. 

Fig. 1. Targeted lipidomics of anandamide metabolism. Postulated pathways of anandamide metabolism. Abbreviations PC, phosphatidylcholine PE, phosphatidylethanolamine NAT, JV-acyl transferase LPA, lysophosphatidic acid PA, phosphatidic acid NAPE, jV-acyl-phosphatidylethanolamine Lyso-NAPE, l-lyso,2-acyl-OT-glycero-3-phosphoethanolamine-JV-acyl ABHD-4, a//3 hydrolase-4 GP-anandamide, glycerophospho-anandamide PAEA, phospho-anandamide PLA, phospholipase A NAPE-PLD, NAPE phospholipase D PLC, phospholipase C FAAH, fatty acid amide hydrolase P, phosphatase COX, cyclooxygenase LOX, lipoxygenase CYP450, cytochrome P450 PDE, phosphodiesterase.

In the biochemical method, the enzyme phospholipase A2, isolated from Naja naja snake venom can attack the native alkenylacylglycerophosphocho-line and liberate completely the esterified fatty acid and the alkenyl(lyso)glyc-erophosphocholine. On the basis of the stereospecific mode of attack of this enzyme on the 2-acyl ester position of sn-3 phosphoglycerides, it can be concluded that the naturally occurring alkenylacylglycerophosphocholine possessed the sn-3 stereochemical configuration. 

Phospholipase A2 Action. As in the case of phosphatidylcholine, the above-mentioned phospholipases will attack only the sn-3 form of naturally occurring (as well as synthetic) phosphatidylethanolamine. The products are, of course, lysophosphatidylethanolamine (1 -6>-acyl-2-lyso-.rn-glycero-3-phosphoethanolamine) and the fatty acids (liberated from the sn-2 position). The latter can be analyzed for composition and structure, as the methyl esters, by gas-liquid chromatography coupled with mass spectrometry. Usually these acyl groups are largely the unsaturated types. 

Assuming that the above enzymatic reaction was run in an ether-rich medium, the products can be isolated by thin-layer chromatography. Thus, the fatty acids released from the sn-2 position can be easily recovered as well as the lysophosphatidylethanolamine. The latter derivative will contain the fatty acyl groups associated with the sn-1 ester position. Base-catalyzed metha-nolysis of the lyso compound will produce the methyl esters. In the usual instance, these will contain mainly saturated chains. In any event the attack by phospholipase A2 can proceed smoothly to completion. These results would strongly support an sn-3 stereochemical configuration for the parent diacylphosphatidy lethanolamine. 

HPLC column was directed into a triple quadropole mass spectrometer equipped with an atmospheric pressure articulated ion spray (electrospray) source. Using MS-MS conditions, dissociation of the parent ions yielded daughter ions comparable to the IM+H]+ of PAF and other molecules, such as O-phosphocholine. Quantitative analyses were obtained by selective ion monitoring. Amounts as low as 0.3 ng of PAF and similar compounds were detected. Interestingly with the lyso derivatives, the limited of detection was 3 ng. The primary alkyl chain lengths present in the neutrophil-derived PAF were 16 0 and 18 0. No mention was made of the presence of any 1-0-long chain acyl analogs. 

O-long-chain acyl glycerophosphoric acid [lyso (acyl) PA]... 

About 1 percent of the total phosphoglycerides that occur in animal cells are in the form of lysophosphoglycerides, in which one of the acyl substituents, usually from C-2, is missing. The lysophosphoglycerides are named by adding the prefix lyso- to the name of the parent phosphoglyceride. 

Figure 8 Comparison of NDB- and BODIPY-labeled lyso glucosyl ceramide and NBD-labeled amino acyl glucosyl ceramide (147).

A phospholipid monolayer in the surface is consistent with the current model that LD are formed by TAG deposition between the two leaflets of the ER membrane and may remain connected to it [144, 145 see below]. Distribution of acyl-CoA cholesterol acyltransferase-1, a major enzyme that synthesizes cholesterylester, in the entire ER [148] seems to indicate that LD may bud anywhere along the membrane. However, Cap-LC/ESI mass spectrometry showed that FA moieties of phosphatidylcholine and lyso-phosphatidylchohne in LD are distinct from those in the rough ER [149]. The results do rule out the generation of the LD surface generated from the ER membrane but indicate that the former is a highly differentiated domain. Mature LD might be independent of the ER. Alternatively, the LD may be connected to the ER, but some molecular mechanism may demarcate the LD surface from the bulk ER membrane as postulated for other ER domains [150]. Whatever is true, TAG synthesized in wide areas of the ER do not deposit indiscriminately but are concentrated to loci specialized to make LD. ADRP or other LD-associated proteins may be involved (see below). 

During neuronal stimulation, the activation of multiple phospholipases seems to be the norm and it can be very difficult to sort out the interactions among them. For example, whereas PA generated by PLD is a potent signal transduction molecule, it can be further metabolized to Lyso-PA, another signal transduction molecule with different effects, by the actions of PLA2. Lyso-PA can be converted back to PA by lysophosphafidic acid acyl transferase (LPAAT), and PA can be converted to DAG by phosphatidate phosphohydro-lase. These are just a few examples of the complex interactions that can occur during neuronal activation. 

Figure 6. Biosynthetic pathways for PAF. PAF can be synthesized by two different metabolic pathways. The first pathway (I) is initiated by phospholipase A2 hydrolysis of alkyl-acyl-GPC, followed by acetylation of lyso-PAF. The second pathway (II) acetylates 1-O-alkyl-2- yso-sn-g Ycero-3-phosphate, followed by dephosphorylation to yield 1-0-alkyl-2-acetyl-s/7-glycerol. This moiety subsequently condenses with CDP-choline to produce PAF.

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