Phosphatidic acid products

The first experiments implicating a role for PLD activity in neutrophil function were performed by Cockcroft and colleagues (Cockcroft Stutchfield, 1989 Cockcroft, 1992) who measured phosphatidic acid accumulation in cells whose membrane phospholipids or ATP were radiolabelled. These experiments showed that phosphatidic acid accumulation during cell activation did not derive from DAG, but rather was directly generated from a phospholipid. Phosphatidic acid production from DAG (generated by PLC)... 

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.

Siegel RK. (1979). Ginseng abuse syndrome. Problems with the panacea. JAMA. 241(15) 1614-15. Simon MF, Chap H, Braquet P, Douste-Blazy L. (1987). Effect of BN 52021, a specific antagonist of platelet activating factor (PAF-acether), on calcium movements and phosphatidic acid production induced by PAF-acether in human platelets. Thromb Res. 45(4) 299-309. 

Mauco, G., Chap, H., Simon, M.-F., and Douste-Blazy, X. Y. (1978) Phosphatidic and lyso-phosphatidic acid production in phospholipase C- and thromlin-treated platelets. Possible involvement of a platelet lipase, Biochimie 60, 653-661. 

Bauldry, S.A., Elsey, K.L. and Bass, DA. (1992). Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production. ]. Biol. Chem. 267, 25141-25152. 

PA is a minor component of the ER membrane that accounts for less than 1% of total ER membrane lipids (Allan, 1996). Formed PA is rapidly consumed by the activity of phosphatidate phosphohydrolase (PAP). In order to measure the formation of PA, the dynamics of PA formation and consumption has to be controlled. This is achieved by exploiting a unique transphosphatidylation reaction that is catalyzed by PLD enzymes. In this reaction, the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. In the presence of low concentrations of primary alcohols, PLD enzymes generate phospha-tidylalcohols, which are not recognized by PAP and are not efficiently consumed (Morris et ah, 1997). Therefore the measurement of transphosphatidylation activity of PLD provides a convenient assay that avoids the otherwise highly dynamic nature of the lipid remodeling cascade induced by Sari to support COPII mediated ER export. 

Triacylglycerols arise not by acylation of glycerol itself but by a sequence of steps m which the first stage is acyl transfer to l glycerol 3 phosphate (from reduction of dihy droxyacetone 3 phosphate formed as described m Section 25 21) The product of this stage IS called a phosphatidic acid... 

Phosphatidic acid is glycerol esterified at the sn-1 and sn-2 positions to two fatty acids and at the sn-3 position to phosphoric acid. It is a product of phospholipase D action that is also an intermediate in the biosynthesis of phosphatidylseiine and phosphatidylinositol. 

The rate of production of DAG in the cell does not occur linearly with time, but rather it is biphasic. The first peak is rapid and transient and coincides with the formation of IP3 and the release of Ca2+ this DAG is therefore derived from the PI-PLC catalyzed hydrolysis of phosphatidylinositols [1]. There is then an extended period of enhanced DAG production that is now known to be derived from the more abundant phospholipid phosphatidylcholine (PC), which has a different composition of fatty acid side chains [9]. Although DAG may be generated directly from PC through the action of PC-PLC, it can also be formed indirectly from PC. In this pathway, PC is first hydrolyzed by PLD to give choline and phosphatidic acid, which is then converted to DAG by the action of a phos-phatidic acid phosphatase [10,11 ]. 

Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid. J. Biol. Chem. 263, 8210-14. 

Phospholipid turnover also takes place in an asymmetric manner. The enzymes responsible for phospholipid turnover in response to receptor-mediated phospholipase c activation are active from the cytoplasmic surface of the membrane. Likewise, diacylglycerol kinases converting the product of phospholipase c back into the key intermediate of phospholipid biosynthesis, phosphatidic acid, are also located on the cytoplasmic smface of the membrane (Sanjuan et al., 2001). 

Jones, 1990 Nishizuka 1992 and 1995). The second, more sustained inaease in DAG comes from the hydrolysis of PC by PC-specific PLC (direct-route), or indirectly via the sequmtial activation of phosphohpase D (PLD) and phosphatidic acid phosphohydrolase (PAP) to generate PA and DAG, respectively. The second phase of DAG production proceeds without an elevation in the concentration of intracellular and might be related to the activation of Ca -independent protein kinase C isoforms (Wakelam, 1998). 

This kind of work, although rather significant, has not been repeated, partly because of the difficulty of obtaining the four enzymes with a sufficient degree of purity. Some work has been done with the idea of using liposomes formed by phosphatidic acid (the product of the first two enzymes shown in Figure 11.7), see Luci (2003). 

As already mentioned, early attempts have been focused on the enzymatic production of lecithin in lecithin liposomes (Schmidli et al, 1991). The metabolic pathway was the so-called Salvage pathway, which converts glycerol-3-phosphate to phosphatidic acid, then diacylglycerol and hnally phosphatidylcholine. Production of the cell boundary from within corresponds to autopoiesis and would close the circle between minimal cell and the autopoietic view of cellular life. 

Triacylglycerols are formed by reaction of two molecules of fatty acyl-CoA with glycerol 3-phosphate to form phosphatidic acid this product is dephosphoiylated to a diacylglycerol, then acylated by a third molecule of fatty acyl-CoA to yield a triacylglycerol. 

Definition and example of a glycerophospho-lipid Phospholipids that contain glycerol are called glycerophospholipids or phosphoglyc-erides. All contain phosphatidic acid, the simplest glycerophospholipid. When an alcohol, such as choline, is esterified to phosphatidic acid, the product is phosphatidylcholine. 

Lysophospholipids have been found in butter serum by Cho et al. (1977). They characterized the sn-1 and -2 lysophosphatidylcholines and phosphatidylethanolamines. It is not known if these compounds are products of degradation or remnants of biosynthesis. Cho et al. (1977) searched for, but did not find, another possible product of enzymatic degradation of milk, phosphatidic acid. Phosphatidic acid can be formed by the action of phospholipase D on phosphatidylcholine, for example, but this enzymatic activity was not detected. The compound is also an important intermediate in the biosynthesis of lipids, but the concentration in tissue is always very low. The amount is also low in milk. Cho et al. (1977) found 1.2 and 0.9 (percent of total lipid P) of the lyso compounds above. The quantities of the other phospholipids were phosphatidylethanolamine, 27.3 -choline, 29.1 -serine, 13.4 -inositol, 2.5 and sphingomyelin, 25.6. 

Sigma Chemical Co., St. Louis, MO. Dimyristoyl and distearoyl lecithins (DML and DSL) and synthetic dipalmitoyl phosphatidic acid (DPPA) were products of Applied Science, State College, Pa. 

Amino acids, amino groups, amino sugars, and nucleic acid derivatives usually account for >95% of the organic N in soils (Anderson et al., 1989), and many other N-containing compounds have been reported in trace amounts (Stevenson, 1994). Anderson et al. (1989) have found traces of L-phosphatidic acid, choline, ethanol-amine, and uric acid (the end product of N metabolism of many animals), which can be oxidized to allantoin, cyanuric acid, and urea. 

This experiment is usually run using [3H]ethanol and locating the radioactive product by thin-layer chromatography. An alternative method would be to label the cells with [3H]oleic acid and [32P]phosphate and then monitor the ratio of the tritium label to phosphorus-32 label in any detectable phosphatidic acid and compare to the parent phosphoglycerides. The ratio would not change if the phosphatidic acid were derived by action of phospholipase D in the stimulated cells. However, the assay of choice at the present time is that associated with the formation of phosphatidylethanol. 

Phospholipase D. This enzyme will attack phosphatidylserine with the liberation of serine and formation of phosphatidic acid. The methodology is exactly the same as the one outlined in Chapter 4. The source of enzyme can be Streptomyces chromofuscus or cabbage, and products of its action are recovered in a chloroform-soluble and a water-soluble fraction. All of the lipid P should be in the chloroform-soluble fraction, and all of the serine should be in the water-soluble fraction. The phosphatidic acid can be identified by its thin-layer chromatographic behavior and its fast atom bombardment-mass spectrometric pattern. Serine can be identified by the procedures outlined earlier. 

During hormonal stimulation of Ptdlns 4,5-P2 hydrolysis there appears to be a preferential degradation of molecules, such as diacylglycerol and phosphatidic acid, which contain arachidonate in the 2-position. Two separate pathways have been proposed for the release of arachidonic acid from these two products of the phosphoinositide response. The first proposes that diacylglycerol is the source of the liberated arachidonate and that diacylglycerol lipase acts on the DG released by hydrolysis of phosphoinositides. The second suggests that a phosphatidic acid-specific phospholipase A2 is responsible for cleaving the arachidonic acid from phosphati-date. 

Gj, G0, Gq). As a result, the inner leaflet of the plasma membrane is the source of a variety of chemical mediators that are released as a consequence of receptor activation. These mediators include inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), which are both products of Pi-specific phospholipase C (Fig. 6-23) and arachidonic acid, which is an unsaturated fatty acid product of phospholipase A2 and phosphatidate (a product of phospholipase D). IP3 induces the release of Ca2+ ions from intracellular endoplasmic reticulum stores. DAG is a known activator of a lipid-dependent serine/threonine protein kinase (protein kinase C). 

The stimulation of phosphatidic acid phosphatase by ethanol stimulates the production of diacylglycerol which in turn stimulates the synthesis of triacylglycerol. Therefore, the triacylglycerol concentration increases in the liver cells. 

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