Phospholipase sources

Encompassing approx 6000 medicinal plant species, the medicinal flora of Asia and the Pacific comprise a fantastic source of pharmacologically active products, and the number of plant species principally used for the treatment of inflammation can be estimated to be more that 380. This chapter will focus on the potentials of medicinal plants of Asia as a source of original anti-inflammatory drugs, with particular interest payed to inhibitors of phospholipase A2, COX, lipoxygenases, elastase, and NOS. 

Classical examples of anti-inflammatory Asteraceae are Arnica montana and Calendula officinalis, both used in European medicine to treat bruises and contusions. There is an expanding body of evidences to suggest that Asteraceae could be a useful source of anti-inflammatories, such as sesquiterpene lactones and/or triterpene alcohols, the latter being known to inhibit 12-O-tetra-decanoylphorbol-13-acetate (TPA)-induced inflammation in mice as efficiently as commercial indomethacine by possible inhibition of phospholipase A2 (10). 

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 6.20. Role of phospholipase D in NADPH oxidase activation. In (a) neimophils were preincubated with [3H]-alkyl-lyso-PAF (5 /iCi/ml) for 60 nun at 37 C. The cells were then washed twice with RPMI 1640 medium and finally resuspended at 2 x 10 cells/ ml in the presence ( ) and absence ( ) of 100 mM ethanol. The cells were then stimulated with 1 pM fMet-Leu-Phe and, at time intervals,aliquots were removed for analysis ofphos-phatidic acid ( ) and phosphatidylethanol ( ) by thin layer chromatography (TLC). In (b), neutrophils were incubated in the presence and absence of 10 mM butanol, and luminol chemiluminescence (10 jUM, final concentration of luminol) was measured after stimulation by 1 jUM fMet-Leu-Phe. Source Experiment of Gordon Lowe and Fiona Watson.

Figure 6.21. Role of phospholipase D in receptor up-regulation. Neutrophils were incubated in the presence or absence of fMet-Leu-Phe and butanol for 15 min prior to analysis of expression of CDllb by FACS analysis. In (a), neutrophils were not stimulated and suspensions did not contain butanol. In (b), suspensions did not contain butanol, but were stimulated with fMet-Leu-Phe. The hatched lines show the receptor expression of suspensions incubated with 10, 20 and 30 mM butanol for 5 min prior to stimulation by fMet-Leu-Phe. Source Experiment of Fiona Watson.

Eurther work on the isolation of related compounds from mammalian sources, which spanned several decades, led to the identification of a large group of structurally related substances. Investigations on their biosynthesis made it evident that aU eventually arise from the oxidation of the endogenous substance, arachidonic acid. The individual products induce a variety of very potent biological responses, with inflammation predominating. Arachidonic acid, once freed from lipids by the enzyme phospholipase A2, can enter one of two branches of the arachidonic acid... 

A very satisfactory source of phospholipase A2 is the venom of the snake, Crotalus adamanteus (Eastern diamondback rattlesnake). This venom can be obtained in lyophilized form from commercial suppliers such as Miami Ser-pentarium (Miami, FL). Of importance, the lyophilization process does not alter the chemical, physical, or enzymatic characteristics of the original venom obtained from this snake. 

In contrast to the phospholipase C of bacterial origin, there appears to be a consensus that the enzyme from mammalian sources prefers Ca2+ at millimo-lar concentrations. Interestingly, heavy metal ions such as Hg2+ or Zn2+ are strong inhibitors of this source of enzyme and EGTA must be included in the reaction mixture to chelate these cations. 

An acetone powder of Savoy cabbage leaves (prepared by heat coagulation and acetone precipitation and available from commercial sources) is suspended in diethyl ether and then filtered. The residue is dispersed in sodium acetate and calcium chloride at pH 5.0, and the insoluble matter is separated by centrifugation at 13,000 g for 10 min at 4°C. The supernatant contains all the phospholipase D activity and in our laboratory s experience can be used successfully in any experiment requiring this enzymatic activity. It can be maintained at -20°C for 1-2 weeks. 

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). 

In another detailed study on the influence of the substituent at the C-l position in sn-3 choline phosphoglycerides, Waku and Nakazawa (1972) investigated the catalytic behavior of the phospholipase D of cabbage and carrot. Their findings showed that either source exhibited high activity toward a diacylphosphoglyceride, with a much lower rate of hydrolysis toward an alkylacyl- and alkenylacylphosphoglyceride. 

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. 

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