1,3-Diacylglycero-2-phosphocholines--synthesis, aggregation behaviour and properties as inhibitors of phospholipase D.

A series of 1,3-diacylglycero-2-phosphocholines (1,3-PCs) with acyl chain lengths of C8-C18 were synthesised by chemical introduction of the phosphocholine moiety into the regioisomerically pure 1,3-diacylglycerols, which were obtained from glycerol and the vinyl esters of fatty acid by means of lipase from Rhizomucor mihei. The 1,3-PCs being regioisomers of the natural glycerophospholipids were studied with respect to their aggregation behaviour in the absence and in the presence of sodium dodecylsulfate (SDS) as well as their properties as substrates and inhibitors of phospholipase D (PLD) from cabbage. While the main structures of the pure 1,3-PCs were micelles (C8), liposomes (C10, C12) or planar bilayers (C14, C16, C18), the addition of SDS resulted in the formation of mixed micelles (C8, C10) and mixed liposomes (C12, C14, C16, C18). None of the 1,3-PCs was found to be hydrolysed by PLD, whereas all of them showed inhibitory properties in the standard assay for PLD. The inhibitory power was strongest with 1,3-didecanoylglycero-2-phosphocholine (IC50 = 43 microM).

Hexadecylphosphocholine and 2-modified 1,3-diacylglycerols as effectors of phospholipase D.

The kinetic behaviour of phospholipase D (PLD) from cabbage has been studied in the presence of several substrate-like compounds such as hexadecylphosphocholine (HPC) and 1,3-didodecanoylglycero-2-phosphatides. 1,3-Didodecanoyl- glycero-2-phosphocholine (1,3-DiC12PC) was found being not cleft by PLD, whereas HPC is hydrolyzed by PLD with small rate. The plot of initial velocity vs. substrate concentration for HPC is more sigmoidal than those for the common substrate phosphatidylcholine (PC)/sodium dodecylsulfate (SDS) (1:0.5) or the short-chain 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DiC6PC). The anionic amphiphiles 1,3-didodecanoylglycero-2-sulfate and 1,3-didodecanoylglycero-2-phosphate act as activators of PLD towards PC similar to SDS. In contrast, 1,3-DiC12PC shows inhibitory properties with an increase in the sigmoidicity of the initial velocity as a function of substrate concentration in the PC/SDS assay. Also HPC inhibits the hydrolysis of PC/SDS, whereas it acts as activator or inhibitor in the hydrolysis of DiC6PC. The results suggest that PLD possesses two substrate-binding sites, where one binds substrate in function of an effector without catalytic activity while the other is the catalytic site. HPC and 1,3-DiC12PC are assumed to compete with the substrate for both binding sites with effects depending on the ratio of concentrations and affinities of substrates and effectors.

2-modified 1,3-diacylglycerols as new surfactants for the formation of reverse micelles.

New 2-modified 1,3-diacylglycerols such as 1,3-dilauroylglycerol 2-disodiumphosphate and analogues were characterized with respect to their tendency to form reverse micelles in isooctane and isooctane/1-hexanol. The water content of the reverse micelles was determined by Karl-Fischer titration. The critical micelle concentration of the compounds was estimated by fluorescence measurements using rhodamine B as indicator. The concentration regions where reverse micelles are observed were characterized by pseudoternary phase diagrams. The ability of the surfactants to extract proteins into the organic phase was examined for cytochrome c. The properties of the new compounds were compared with the behaviour of the corresponding regioisomeric 3-modified 1,2-diacylglycerols as well as lecithin and the surfactant AOT, which is preferably used for the formation of reverse micelles. The results suggest that the position of the head group in the modified diacylglycerols is of low importance for the phase behaviour whereas the ability to form reverse micelles decisively depends on the kind of the head group.