Resistance to phospholipases

Both the normal and abnormal forms of PrP are derived from the same gene and have the same amino acid sequence. Pulse-chase studies in Sc+-MNB have demonstrated that PrP-res is in fact posttranslationally derived from mature PrP-sen (Borchelt et al., 1990 Caughey and Raymond, 1991). Unlike PrP-sen, PrP-res is resistant to phospholipases and protease treat-... 

Synaptic stimulation, ischemia or seizure activates phospholipase A2 and releases arachidonic and docosahexaenoic acids 578 Secretory phospholipases A2 are of relatively low molecular weight and have a high number of disulfide bridges, making them relatively more resistant to denaturation 579 There are high-affinity receptors that bind secretory phospholipase A2 579... 

To date the evidence seems to favor the binding of tumor promoter to phospholipid in the cell membrane. Specific binding of [3h]TPA to mouse epidermal particulate matter is susceptible to phospholipases C and A2, less susceptible to protease, and completely resistant to glycosidase (32). Photoaffinity labelling studies with [20-3h]-phorbol 12-p-azidobenzoate 13-benzoate indicates that the irreversible binding of this photolabile phorbol ester to mouse brain membrane is predominantly to the phospholipid (specifically phosphatidylethanolamine and phosphatidylserine) portion rather than to the protein portion (33). 

In addition to DPIEL, other phosphatidyhnositol analogues (PIAs) (e.g., SH-5 and SH-6, compounds 36 and 37, respectively. Fig. 7) have been recently reported [ 159,160]. When used at 5 or 10 xM, SH-5 and SH-6, which are supposed to be more resistant to phosphatidyUnositol-specific phospholipase C mediated degradation [161], effectively block the phosphorylation and activation of PKB in HL60AR tumor cells, and sensitize this and other leukemic tumor cell hnes to the effects of etoposide and cytarabine. No effect on the survival rate of hematopoetic precursor cells was observed if the compounds are used at 5 xM. 

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],... 

If any analytical evaluation of the progress of the reaction is wanted, then an aliquot can be diluted with 95% ethanol (or methanol) and titrated with 0.02 N methanolic NaOH with cresol red as the endpoint indicator. An alternate approach is to employ a titrimeter (Radiometer, microtitration assembly, Copenhagen), but it is important to realize that most phospholipases A2 adhere strongly to glass surfaces. Thus a vessel resistant to the solvents used in the reaction is mandatory, as is rigorous attention to cleaning the electrode after titration. The extent of the enzymatic reaction can be calculated using as a control a reaction mixture with no enzyme added. 

Furthermore, the unnatural -l configuration of the backbone glycerol would impart resistance to attack by phospholipases released by other organisms and would thus have a survival value for the archaebacteria. More specific functions of archaebacterial lipids will now be considered. 

The relative abundance of a particular phospholipid in the two leaflets of a plasma membrane can be determined on the basis of its susceptibility to hydrolysis by phospholipases, enzymes that cleave various bonds in the hydrophilic ends of phospholipids (Figure 5-9). Phospholipids in the cytosolic leaflet are resistant to hydrolysis by phospholipases added to the external medium because the enzymes cannot penetrate to the cytosolic face of the plasma membrane. 

Based mainly on cell-free assays, 10 enzymatic activities that degrade phospholipids, intermediates in the phospholipid biosynthetic pathway, or triacylglycerol have been reported (Table 2). The detergent-resistant phospholipase A, (encoded by pldA) of the outer membrane, characterized by Nojima and colleagues (Y. Nakagawa, 1991), is the most studied of these enzymes. This enzyme is unusually resistant to inactivation by heat and ionic detergents... 

It was generally assumed that oxidation products from oils and fats are poorly absorbed. Indeed, animals can be resistant to the effects of abused oils in their diet (29,30). Nevertheless, orally administered oxidized C-linoleic acid was incorporated into chylomicrons and very low density lipoprotein (VLDL) particles in rats. The absorbed oxidation products were subsequently identified as hydroxy fatty acids (31). Interestingly, dietary glutathione (GSH) reduced the absorption of peroxidized linoleate (32), presumably due to the activity of selenium-dependrait GSH peroxidase present in the intestinal epithelial cell (33). However, these absorption studies used free fatty adds, not triglycerides thus, they cannot address the role of pancreatic lipase and phospholipase. The fate of polymerized fatty adds has not been studied specifically. Whether the intestinal flora could degrade fliese polymers and hence allow their absorption remains to be seen. What is clear is that very little is known about the absorption of oxidized fat in animals and even less in humans. 

Whether oxidants affect the lipase action or promote the synthesis and release of TG-rich lipoproteins has not been determined. It is also possible that TG containing peroxidized fatty acid components are resistant to lipolysis. Earlier studies have demonstrated that antioxidants enhanced the (hepatic) lipase actions [66] however, these studies have not been corroborated or confirmed by more robust studies. In contrast, phospholipases seem to act more robustly on peroxidized or oxidatively tailored phospholipids [67-70] although the enzyme itself could be inactivated by oxidants [70], Recently it was reported that the protein disulfide isomerase, a redox-sensitive enzyme, could contribute to the endoplasmic reticulum-associated degradation of apoB through its chaperone activity [71],... 

Phospholipases Aj (EC 3.1.1.4) hydrolyse the fatty acyl ester bond at the sn-2 position of membrane phospholipids to yield free fatty acids such as ara-chidonic acid. Recognised isoforms include i) low molecular weight, 14 kDa proteins that are inhibited by reducing agents, require calcium for activation and exhibit no preference for the hydrolysis of arachidonic acid vs. other fatty acids ii) cytosolic, an 85 kDa protein that is resistant to reducing agents, calcium-requiring and preferentially hydrolysed arachidonic acid and iii) calcium-indepen-... 

Synthesis of the first generation C-phosphonate glycosylphos-phatidylinositols (GPI) analogs (427) has been reported by Vishwakarma et al. The key step in this protocol involved coupling of a-pseudodi-saccharide (425) with phosphonic acids (426) in quantitative yield (Scheme 129). It has been also demonstrated that these synthetic probes (427) were resistant to hydrolysis by phosphatidylinositol-specific phospholipase C (PI-PLC) and showed moderate inhibition of the enzyme activity. 

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