Hydrolysis with phospholipase

The ozonides of choline and ethanolamine phosphatides subjected to reduction with PhsP yield the corresponding core aldehydes. After hydrolysis with phospholipase C to eliminate the polar group and silylation with trimethylsilyl chloride, the core aldehydes can be determined by GLC-FID using temperature programming to high temperatures . ... 

Glycerophospholipids may be hydrolyzed to phos-phatidic acid with phospholipase D, or to di-acylglycerols with phospholipase C. The latter hydrolysis allows the analysis of phospholipids by TLC, GC, or LC, whereas the hydrolysis with phospholipase D produces molecules that are best analyzed by LC. 

Zhang et al. studied a membrane reactor for the production of ceramide through sphingomyelin hydrolysis with phospholipase C from Clostridium perfringens. Ceramide has aroused considerable interest as an active component in both the pharmaceutical and the cosmetic industry. The enzymatic hydrolysis of sphingomyelin has proved a feasible method for the production... 

The phosphoinositides PIP and PIP2 were further identified by hydrolysis with phospholipase A2, giving products which co-migrated with respective standards. Results are means of 2 determinations from 3 separate experiments. 

Figure 6.7. Phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C. Occupancy of receptors (R) results in exchange of bound GDP for GTP on the a-subunit of a het-erotrimeric G-protein. The a-subunit then dissociates from the fi- and y-subunits and activates phospholipase (PLC). This enzyme is calcium dependent and, upon activation, can hydrolyse phosphatidylinositol 4,5-bisphosphate (PIP2). The products of this hydrolysis are inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which is released into the cytoplasm, and diacylglycerol (DAG), which remains in the membrane. The DAG is an activator of protein kinase C, which moves from the cytoplasm to the membrane, where it forms a quaternary complex with DAG and Ca2+. 

Chemicals such as these bind with phospholipids, inhibiting their hydrolysis by phospholipases. They can also interact with phospholipases, thus limiting the ability of the enzyme to metabolize phospholipid. Drugs, which have this structure, may also influence the synthesis of phospholipids. 

Certain classes of lipids are susceptible to degradation under specific conditions. For example, all ester-linked fatty acids in triacylglycerols, phospholipids, and sterol esters are released by mild acid or alkaline treatment, and somewhat harsher hydrolysis conditions release amide-bound fatty acids from sphingolipids. Enzymes that specifically hydrolyze certain lipids are also useful in the determination of lipid structure. Phospholipases A, C, and D (Fig. 10-15) each split particular bonds in phospholipids and yield products with characteristic solubilities and chromatographic behaviors. Phospholipase C, for example, releases a water-soluble phosphoryl alcohol (such as phosphocholine from phosphatidylcholine) and a chloroform-soluble diacylglycerol, each of which can be characterized separately to determine the structure of the intact phospholipid. The combination of specific hydrolysis with characterization of the products by thin-layer, gas-liquid, or high-performance liquid chromatography often allows determination of a lipid structure. 

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. 

Enzymatic hydrolysis of the polar head group of a phospholipid molecule can be carried out with phospholipase C and phospholipase D. Phospholipase D is used to exchange the amino head group of phosphatidylcholine with serine to form phos-phatidylserine (164). 

P NMR studies on locust lipophorin indicate that a large proportion of the PLs reside on the surface of lipophorin (Katagiri et al., 1985). In addition to this study, the susceptibility of lipophorin phospholipids to hydrolysis by phospholipase A2 is consistent with a surface localization of PLs (Katagiri et al., 1985 Kawooya et al., 1991). Although the amphi-pathic nature of PLs makes this a logical conclusion, it has to be pointed out that the use of hydrolytic enzymes to demonstrate the location of any component would be valid only if a rearrangement of the lipid components in lipophorin does not occur on the time scale of the experiment. Because this condition is never met, a cautious interpretation of such data is necessary. 

In 1955 Blomstrand [93] fed phospholipids labeled with [ " C]palmitic acid to rats with both the bile and thoracic duct cannulated. The results indicated that in this situation 68% of the fatty acids were absorbed and that bile was not obligatory for absorption of phospholipid fatty acid. The distribution of the labeled fatty acid in the thoracic duct lipids showed the same pattern as after feeding the free fatty acid, indicating that the phospholipids had been hydrolyzed before absorption. The mechanism responsible for the hydrolysis of phospholipid in bile fistula rats is not obvious. A novel enzyme with phospholipase Aj activity has been demonstrated in the rat intestine [94]. This enzyme has its highest specificity for phosphatidyl glycerol and its role for the hydrolysis of dietary PC is unclear. 

The ability of PI synthetase to use 5-deoxy-5-fluoro-myo-inositol (4) as a substrate was confirmed by use of a radiolabeled compounds as shown in Figure 7. PI synthetase incorporated the analog into lipid in a time-dependent manner. The incorporation was absolutely dependent on the presence of CDP-diglyceride and was inhibited by the presence of myo-inositol (1) in the incubation mixture, as expected for PI synthetase. Chromatography of the reaction mixture revealed that a single radiolabeled product was formed with a mobility similar to, but distinct from, that of PI. Subsequent analysis has shown that the product is converted to a water-soluble form on mild alkaline hydrolysis and yields 5-deoxy-5-fluoro-myo-inositol (4) on treatment with phospholipase D, in agreement with the formation of phosphatidyl-5-deoxy-5-fluoro-myo-inositol as the product (data not shown). Determination of the absolute structure of these phospholipids awaits large-scale enzymatic synthesis, isolation of the product, and studies by mass spectrometry and NMR spectroscopy. 

The mechanism of an increased phosphatidylinositol metabolism has been studied in platelets stimulated with thrombin in some detail. Thrombin stimulation leads to formation of the 1,2-diacylglyceride and inositol phosphate through hydrolysis by phospholipase C (Fig. 4). According to Majerus and coworkers, the diacylglyceride is then hydrolysed by a diglyceride lipase with liberation of arachidonic acid [35,40]. In contrast, Lapetina and his colleagues [41,42] propose that the diacylglyceride is phosphorylated to phosphatidic acid, a likely calcium ionophore [43], which in turn... 

The bioactive core aldehydes derived from the oxidation of LDL phospholipids (see Section E.2) can be analysed either directly by LC-MS or after hydrolysis by phospholipases and sodium borohydride reduction. These aldehyde esters are purified by solid-phase extraction followed by preparative TLC. Unwanted ester containing phospholipids can be removed after treatment with phospholipase Al. The more polar oxidized phospholipids are eluted by HPLC after the class of phospholipids from which they are derived. The core aldehydes derived from oxidized phospholipids (Section E.2) are also known to be pro-inflammatory. Only a few C-4 and C-5 core aldehydes have been identified and analysed by LC-MS, either directly or after derivatization in oxidized LDL. Many more saturated and unsaturated core aldehydes can be expected in oxidized LDL, according to the degree and mode of oxidation. However, the abihty of these core aldehydes to form covalent adducts with the apoB of LDL may prevent them from being detected directly by LC-MS. Chemical or enzymatic hydrolysis may therefore be required before they can be analysed by this technique. 

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