Phospholipase Subject

Methods used to demonstrate the existence of membrane phospholipid asymmetry, such as chemical labelling and susceptibility to hydrolysis or modification by phospholipases and other enzymes, are rmsuitable for dynamic studies because the rates of chemical and biochemical reactions are of a different order compared to the transmembrane translocahon of the phospholipids. Indirect methods have therefore been developed to measure the translocation rate which are consequent on the loss of membrane phospholipid asymmetry. Thus time scales appropriate to rates of lipid scrambling under resting conditions or when the forces preserving the asymmetric phospholipid distribution are disturbed can be monitored. Generally the methods rely on detecting the appearance of phosphatidylserine on the surface of cells. Methods of demonstrating Upid translocation in mammalian cells has been the subject of a recent review (Bevers etal., 1999). 

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 . ... 

The marine natural product ascidiatrienolide A 220 is a strong inhibitor of phospholipase A2. Compound 220 and the closely related didemnilactones 221-223 feature a common hydroxy-substituted anti to the ring oxygen) (Z)-nonenolide core. Lactone 219, which constituted the key intermediate in a previous total synthesis of 220 and can also be elaborated to lactones 221-223, has been the subject of an interesting study by Flirstner s group, that revealed once more the very subtle and cooperative influence of different parameters on the stereochemical course of metathesis reactions. Thus, it was shown that the ElZ ratio obtained in an RCM step is not only dependent on the relative configuration of the cyclization substrate, but also on the chosen catalyst (Scheme 41). When applied to the... 

Phospholipase C, which occurs in different subtypes in the cell, is a key enzyme of phosphatide inositol metabohsm (for cleavage specificity, see Fig. 5.24). Two central signaling pathways regulate phosphohpase C activity of the cell in a positive way (Fig. 6.4). Phospholipases of type CP (PL-CP) are activated by G-proteins and are thus linked into signal pathways starting from G-protein-coupled receptors. Phosphohpases of type Y (PL-Cy), in contrast, are activated by transmembrane receptors with intrinsic or associated tyrosine kinase activity (see Chapter 8, Chapter 10). The nature of the extracellular stimuli activated by the two major reaction pathways is very diverse (see Fig 6.4), which is why the phosphohpase C activity of the cell is subject to multiple regulation. 

A detailed analysis of the effect of mixed monolayers of 15 and DMPC on the activity of phospholipase A2 was reported by Grainger et al. [53]. Monolayers composed of different ratios of DMPC and either 15 or primarily poly 5 were characterized by Langmuir isotherms and isobars. The phospholipse-A2-mediated hydrolysis of selected monolayer compositions was usefully employed to ascertain the effectiveness of the enzyme. Both 15 and polyl5 were resistant to hydrolysis. The DMPC hydrolysis was sensitive to its molecular environment in a manner that suggests the phase separation of the polyl5 from DMPC. Phospholipase A2 activity is known to be sensitive to the concentration of the hydrolytic products, i.e. the fatty acid and lysophospholipid. The effect of these reaction products of the activity of phospholipase A2 on mixed monolayers of nonpolymerizable lipids is the subject of a series of interesting studies which are beyond the scope of this review. Ahlers et al. reviewed some of this research [54],... 

There has been an enormous amount of information published on the isolation, purification, and characterization of the phospholipases, particularly phospholipase A2. Instead of relating a number of these observations, the reader is strongly encouraged to consult an excellent treatise on this subject matter by Waite (1987). Consequently only that information pertinent to the objectives of this section, concerned mainly with the biochemistry of the venom phospholipase A2, will be discussed here. 

Using a physical-chemical protocol, Cymerman-Craig et al. (1966) employed phospholipase C attack on the naturally occurring alkenylacylglycero-phosphocholine to yield alkenylacylglycerol. The latter derivative was subjected to base-catalyzed methanolysis to produce alkenylglycerol. The latter... 

In order to conduct research studies on this novel lipid mediator, it is necessary to have available a sufficient quantity of purified, well-defined material. This has not been an easy task, but an approach to preparation of sphingosine-l-P in milligram amounts was outlined by Van Veldhoven et al. (1989). In this procedure, a commercial preparation of sphingosylphospho-choline (which contains a free amine group due to removal of a fatty acyl residue from initial substrate, sphingomyelin) is incubated with phospholipase D in an ammonium acetate buffer at pH 8.0 for 1 hr. An insoluble reaction product is collected and subjected to purification by dissolution in water at room temperature followed by cooling to 4°C. A precipitate forms again and is collected and treated in a similar manner as above except that acetone is used as the solvent. 

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