Phosphatidate, phosphatidic acid soluble

Fig. 23 Saliva and serum IgA (primary and secondary) response following orally administered soluble antigen Streptococcus mutans cell wall extract (open circles, soluble antigen solid circles, liposome-encapsulated material) (phosphatidylcholine, phosphatidic acid, cholesterol). (From Ref. 277).

These approaches have been used to show conclusively that the initial, low formation of DAG that occurs during activation with soluble agonists comes from PLC activity, and that the later, more sustained generation of DAG comes from PLD activity. Such experiments have also shown that primary alcohols can inhibit the activity of the NADPH oxidase under some conditions. When neutrophils are pretreated with cytochalasin B, primary alcohols are potent inhibitors of 02" secretion, and the kinetics of phosphatidic acid formation are rapid, peaking within about 20 s and coinciding with oxidase activation. However, in the absence of cytochalasin B, primary alcohols have little effect on the initiation of O2" secretion, but decrease the duration of oxidase activity they also inhibit the later phase of luminol chemiluminescence, which is largely intracellular, and the kinetics of phosphatidic acid formation closely parallel the kinetics of this intracellular oxidase activity (Fig. 6.20). Thus, in cytochalasin-treated cells, PLD is activated rapidly, and this activation is required for 02" secretion in the absence of cytochalasin, PLD is activated more slowly and its function is not for the activation of the oxidase, but rather for sustained (and intracellular) activity. 

The lower, chloroform-rich phase is separated carefully from the protein-containing interface, and then it is washed twice with methanol-water (10 9, v/v) and the washes are discarded. The chloroform layer contains the phosphatidic acid (as a sodium salt) and can be isolated by acetone precipitation. The yields can be of the order of 90-95%. One alternative route to identification of the chloroform-soluble material is to analyze it for total phosphorus and total fatty acid ester (see procedures described earlier). In the case of diacylphosphatidylcholine as the substrate, the fatty acid ester/P molar ratio should be 2.0. Another approach is to subject the chloroform-soluble fraction to preparative thin-layer chromatography on silica gel H (calcium ion free) in a two-dimensional system with a solvent system of chloroform-methanol-28% ammonium hydroxide (65 35 6, v/v) in the first direction and a solvent system of chloroform-acetone-methanol-glacial acetic acid-water (4.5 2 1 1.3 0.5, v/v) in the second direction. The phosphatidic acid will not migrate far in the basic solvent Rf 0.10) and will show an Rf value one-half of that of any remaining starting substrate (fyO.40) in the second solvent. Of course with a simple substrate system, one can use the basic solvent in one dimension only... 

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. 

It is a complex mixture of acetone-insoluble phosphatides that consists chiefly of phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol combined with various amounts of other substances such as triglycerides, fatty acids, and carbohydrates. Refined grades of Lecithin may contain any of these components in varying proportions and combinations depending on the type of fractionation used. In its oil-free form, the preponderance of triglycerides and fatty acids is removed and the product contains 90% or more of phosphatides representing ah or certain fractions of the total phosphatide complex. Edible diluents, such as cocoa butter and vegetable oils, often replace soybean oil to improve functional and flavor characteristics. Lecithin is only partially soluble in water, but it readily hydrates to form emulsions. The oil-free phosphatides are soluble in fatty acids, but they are practically insoluble in fixed oils. When ah phosphatide fractions are present, Lecithin is partially soluble in alcohol and practically insoluble in acetone. 

In the bacterial PI-PLC structures, the top of the barrel rim has several hydrophobic residues that are fully exposed to solvent and poorly defined in the crystal structures (implying significant mobility). The active site of PI-PLC is accessible and well-hydrated, and these mobile elements at the top of the barrel offer a different motif for interactions of the protein with phospholipid interfaces. The PI-PLC from B. thuringiensis (nearly identical in sequence to the enzyme from B. cereus whose crystal structure was determined) exhibits the property of interfacial activation, where enhanced activity is observed when the substrate PI is present in an interface compared to monomeric substrate (Lewis et al., 1993). However, other non-substrate lipids such as phosphatidylcholine (PC), phosphatidic acid (PA), and other anionic lipids have an effect on the activity of PI-PLC toward both substrates PI and water-soluble cIP (Zhou et al., 1997). In particular, the presence of PC enhances the catalytic activity of... 

Cmde fractions can be obtained by solvent fractionation treatments utilizing lower alcohols, such as ethanol, or alcohol-water mixtures. The product is a soluble fraction rich in phosphatidylcholine, whereas phosphatidic acid and phophatidyli-nositol predominate in the insoluble fraction. The shift in the ratio of phosphatidylcholine to phosphatidylethanolamine improves the emulsification and antispattering capabilities of the soluble fraction. The products of this process can be used as they are or can be further purified with adsorbents. The soluble fraction is an excellent oil-in-water emulsifier and is predominately used in margarine. The acidic phospholipids of the insoluble fraction are used in water-in-oil systems. The chocolate manufacturing industry uses this fraction to increase the viscosity of chocolate masses, thereby reducing the requirement for cocoa butter (83, 84). 

Another aspect of heating soybeans in particular is the impact on the phospholipase enzyme. The phospholipase enzyme is activated at approximately 55°C and remains activated up to approximately 100°C. In this temperature range, and with sufficient exposed surface area and time, the phospholipase enzyme modifies a portion of the phospatides in the oil fraction by splitting off the non-fatty acid moiety (16). The resultant calcium and magnesium salts of phosphatidic acids that are formed tend to be more oil-soluble than water-soluble, thereby converting phospatides from a hydratable form to a nonhydratable form (16). This has a resultant impact on the quantities of acid, caustic and silica needed to reduce the phosphorus content of the soybean oil in the downstream degumming and refining unit operations. 

The linkage of the mj/o-inositol to the phosphatidic acid has not yet been determined with certainty the inositolphosphoric acid, isolated after hydrolysis of the intact phosphatide (or of the water-soluble portion, obtained by alkaline hydrolysis), gives, on paper chromatography, a mixture of inositol 1(3)-phosphate and a small proportion of inositol 2-phosphate (separated on paper by the method of Pizer and Ballou ). This indicates that, in the phospholipid, the m /o-inositol is probably linked by its C-1- or C-3-hydroxyl group to phosphoric acid. 

In the plastids, acyltransferases provide a direct route for entrance of acyl groups from ACP to membrane lipids. Since this is the standard pathway for phosphatidic acid synthesis in E. coli and cyanobacteria, both the enzymes of phosphatidic acid synthesis in plastids and the glycerolipid backbones they produce are termed prokaryotic . In both chloroplasts and non-green plastids, the glycerol-3-phosphate acyltransferase is a soluble enzyme that, unlike the E. coli enzyme, shows preference for 18 1-ACP over 16 0-ACP. The lysophosphatidic acid acyltransferase, which is a component of the inner envelope of plastids, is extremely selective for 16 0-ACP. The presence of a 16-carbon fatty acid at the... 

Figure 3. The effect of phospholipid head group on the protonation dynamics of an indicator adsorbed on a surface of neutral micelle. The indicator, bromocresol green, was adsorbed on Brij-58 micelles at a ratio of one per micelle. The pulse protonation, measured at pH 7.3, was initiated by photoexcitation of a water-soluble proton emitter 2-naphthol-3,6-disulfonate (2 mM). The reaction was followed spectrophotometrically. A, control no phospholipids added. B, phosphatidylcholine added to amount of 6 molecules per micelle. C, phosphatidyl-serine added to 6 molecules per micelle. D, phosphatidic acid added to 6 molecules per micelle. For more details see Nachliel and Gutman (ll).

A membrane-derived a-amylase was solubilized from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-a-amylase-protein A-Sepharose column. Membrane-derived a-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulphate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform-methanol. The extracted phospholipid was predominantly phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound a-amylase was latent in membrane vesicles. Release of membrane-bound a-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and di-isopropyl fluorophosphate and was stimulated by Ca and Mg. The amount of membrane-bound a-amylase was related to the level of secretion. 

The first hurtle is to reproducibly extract lipids from a matrix. The most common lipids extraction methods are those of Bligh and Dyer [42] and Eolch [43]. Recent analysis of these two methods has shown that the Eolch method tends to have a greater total recovery of lipid [44]. A variety of other solvent mixtures has been compared and may offer fewer hazards with similar recoveries [45]. These extraction methods are designed to recover the principal lipid classes, but may not be as useful for recovery of lipids that have unique charge characteristics. For example, fatty acids, phosphatidic acids, and lyso-phosphatidic acids usually require acidic solvents to facilitate recovery from an aqueous solution while neutral lipids may not be sufficiently soluble in an organic solvent [46]. Other complexities include solvent manipulations required to extract more polar lipids like the phosphatidylinositol phosphates. 

At present, little is known about the phosphatidate phosphohydrolase step in the biosynthetic pathway to triacylglycerols. The enzyme was specifically measured by Moore et al, (1973) in subcellular fractions of castor bean endosperm and found to be located mainly in the endoplasmic reticulum, with some activity in the soluble fraction. In leaves, the highest specific activity was found in an undefined particulate fraction, whereas the m or part of the activity was soluble (Heinz, 1977). Other evidence for a specific phosphatidate phosphohydrolase activity in chloroplast membranes was indirect (Joyard and Douce, 1977). Care must always be taken to ensure that the activity is not due to a nonspecific phosphatase in view of the report by Blank and Snyder (1970) that wheat germ contains an acid phosphatase capable of dephosphorylating phosphatidic acid. The enzyme described by Moore et al. 

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