Phospholipase D enzyme

At present hydrolysis with alkali or phospholipase D enzyme is used to cleave the As moiety in lipid extracts and the water-soluble As species liberated are analyzed by HPLC-ICP-MS [32, 33, 99]. Lipid-soluble As species such as phosphatidyl AC and phosphatidyl arsenosugar on treatment release DMA, MA, AC, and glycerol arsenosugar. 

Figure 2.17 Phospholipid Synthesis. Starting from L-a-glycerophosphocholine CdCla, fatty acids are coupled to free alcohol functional groups by means of carbonyl diimidazole (CDI) yielding a phospho-choline (PC) product. Head group exchange is made possible with Phospholipase D enzyme.

Therefore, it is currently believed that anandamide is formed from membrane phospholipids (Fig. 4) through a pathway that involves (1) a trans-acylation of the amino group of phosphatidylethanolamine with arachidonate from the sn-1 position of phosphatidylcholine and (2) a D-type phosphodiesterase activity on the resulting A-arachidonylphosphati-dylethanolamide (NAPE). Synthesis of anandamide is presumably regulated at the levels of both enzymes, the A-acyltranferase and the phospholipase D, by stimuli that raise intracellular calcium or by receptors linked with cAMP and PKA. It has been shown that anandamide is formed when neurons are depolarized and, therefore, the intracellular calcium ion levels are elevated (Cadas, 1996). 

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.

Synthesizing Enzymes Phospholipase D Phospholipase C Diacylglycerol lipase... 

Anandamide is believed to be synthesized from a phospholipid precursor, /V-arachidonoyl-phosphatidylethanolamine, catalysed by phospholipase D (Di Marzo et al. 1998). The other proposed route of synthesis is from condensation of arachidonic acid and ethanolamine, although this has yet to be demonstrated in living cells. 2-AG is formed in a calcium-dependent manner, and mediated by the enzymes phospholipase C and diacylglycerol lipase (Kondo et al. 1998 Stella et al. 1997). 

A12. Attwood, D., Graham, A. B., and Wood, G. C., The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase-inactivated enzyme by phospholipids and detergents. Biochem. J. 123, 875-882... 

Phospholipase D Cabbage Triton X-lOO/phosphotidyl choline/diethyl ether Enzyme characteristics [87]... 

Phospholipases are enzymes that cleave phospholipids. Phosphohpases of type Al, A2, C and D are differentiated according to the specificity of the attack point on the phospholipid. The bonds cleaved by these phospholipases are shown in Fig. 5.24a. 

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... 

Enzymes that degrade phospholipids are called phospholipases. They are classified according to the bond cleaved in a phospholipid (fig. 19.11). Phospholipases Al and A2 selec- tively remove fatty acids from the sn-1 and sn-2 positions, i respectively. Phospholipase C cleaves between glycerol and the phosphate moieties phospholipase D hydrolyzes the [ head-group moiety X from the phospholipid. Lysophospho- lipids, which lack a fatty acid at the sn-1 or sn-2 position, j are degraded by lysophospholipases. f Phospholipases are found in all types of cells and in... 

Schiavo G, Papini E, Genna G, Montecucco C (1990) An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin. Infect Immun 58 4136 11 Scott AB, Magoon EH, McNeer KW, Stager DR (1989) Botulinum treatment of strabismus in children. Trans Am Ophthalmol Soc 87 174-180 discussion 180 1 Scott D (1997) Phospholipase A2 structure and catalytic properties. In Kini R (ed) Venom phospholipase A2 enzymes structure, function and mechanism. John Wiley Sons, Chichester, p 97-128. 

Finally, though only a direct chemical hyrolysis was described above for liberation of choline, it is possible to employ the enzyme phospholipase D to release choline from intact phosphatidylcholine with the concomitant formation of phosphatidic acid. 

Proof for the existence of phospholipase D in nature (i.e., in plant tissues) was provided by Hanahan and Chaikoff (1947). Subsequently, this enzyme has been detected in microorganism and mammalian cells. An overall, in-depth treatment on phospholipase D is given by Waite (1987). 

Phosphodiesterase (Hydrolysis) Activity. A rather broad substrate specificity is exhibited by the purified phospholipase D (phosphodiesterase activity). It can attack phosphatidylcholine, phosphatidylethanolamine, phospha-tidylserine, and phosphatidylglycerol. In most cases, Ca2+ was an activator, but variable results were obtained on the positive influence of diethyl ether on the catalytic activity of different sources of this enzyme. Usually the optimum pH was in the range from 5.0 to 7.0. Mammalian phospholipase D, containing both the phosphodiesterase and transphosphatidylase activities, exhibited a broad-range substrate specificity similar to that of the plant enzyme. However, the mammalian enzyme showed a dependency for the presence of oleic acid in the reaction system (Kobayashi and Kanfer, 1991). 

It appears on the basis of the above studies that phospholipase D does exhibit a stereopreference and also has preference for a phosphoglyceride with a particular substituent on the C-l position. However, further definition of the behavior of phospholipase D requires the use of highly purified enzyme. 

Phospholipase D Behavior. While this enzyme shows no stereospecificity in its attack on the ethanolamine phosphoglycerides, its action in releasing free ethanolamine and a phosphatidic acid provides very clear evidence that the ethanolamine was attached to the parent molecule via a phosphate ester bond. Again, the same protocols and enzyme sources illustrated in Chapter 4 for phosphatidylcholine can be applied very easily to the ethanolamine phosphoglycerides. 

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. 

Stead, D. 1989. Assay methods for lipases and phospholipases. In Enzymes of Psychrotrophs in Raw Food (R.C. McKellar, ed.), pp. 173-187, CRC Press, Boca Raton, FL. 

---