Monomeric phospholipids, micellization

V. Critical Micelle Concentration Determinations and Micellization of Monomeric Phospholipids by Detergents... 

Similarly, incorporation of monomeric phospholipids by detergents into mixed micelles results in a chemical-shift change in P NMR, and this can be used to follow the micellization process. This is illustrated in Fig. 8, where the micellization of dihexanoyl-PC by titration with Triton X-lOO was followed by changes in the P-NMR chemical shift. In contrast, with the same amount of Triton X-100, dibutyryl-PC is hardly micellized at all. A control titration of the completely water-soluble analog glycerophosphoryl-choline is also included. For the dihexanoyl-PC, a partition coefficient between Triton X-100 micelles and free solution was calculated as a function of the Triton X-100 concentration, using the phase-separation approximation for mixed-micelle formation and considering the mixed micelles to be a pseudo-phase. With the data in Fig. 8, the fraction of dihexanoyl-PC... 

Figure 1 Schematic structures of micelle and liposome, their formation and loading with a contrast agent, (a) A micelle is formed spontaneously in aqueous media from an amphiphilic compound (1) that consists of distinct hydrophilic (2) and hydrophobic (3) moieties. Hydrophobic moieties form the micelle core (4). Contrast agent (asterisk gamma- or MR-active metal-loaded chelating group, or heavy element, such as iodine or bromine) can be directly coupled to the hydrophobic moiety within the micelle core (5), or incorporated into the micelle as an individual monomeric (6) or polymeric (7) amphiphilic unit, (b) A liposome can be prepared from individual phospholipid molecules (1) that consists of a bilayered membrane (2) and internal aqueous compartment (3). Contrast agent (asterisk) can be entrapped in the inner water space of the liposome as a soluble entity (4) or incorporated into the liposome membrane as a part of monomeric (5) or polymeric (6) amphiphilic unit (similar to that in case of micelle). Additionally, liposomes can be sterically protected by amphiphilic derivatization with PEG or PEG-like polymer (7) [1].

We have now extended these studies to synthetic phospholipids that contain short chain fatty acyl groups and which are water soluble, such as dibutyryl and dihexanoyl phosphatidylcholine (PC). These phospholipids are monomeric below their critical micelle concentration (cmc), yet activate the enzyme. In order to carry out kinetic studies, the long chain phospholipid substrate must generally be solubilized by a detergent such as Triton X-100 which serves as an inert matrix. Further understanding of the mechanism of the activation by short-chain phospholipids requires first a quantitation of the solubilization of these compounds by detergent ... 

We found the 31p-NMR chemical shift of monomeric dihexanoyl PC increases upon the addition of the nonionic detergent Triton X-100. This phenomenon was used to quantitate the solubilization of this phospholipid by the detergent micelles as a function of detergent concentration using a simple phase separation model ( 5). Similar studies were carried out on dibutyryl PC. At a phospholipid concentration of 7 mM and 56 mM detergent, 85% of the dihexanoyl PC, but only 3% of the dibutyryl PC was incorporated into the micelles. 

Having shown that dibutyryl PC is monomeric under the enzyme assay conditions, we found that the phospholipase A2, which acts poorly on PE in mixed micelles, is activated by dibutyryl PC which is itself an even poorer substrate. 31p-NMR spectroscopy was employed to show that only PE is hydrolyzed in mixtures of various compositions of these two phospholipids. The fully activated enzyme hydrolyzes PE at a similar rate to its optimal substrate, PC containing long-chain fatty acid groups. Because dibutyryl PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzyme by phosphoryl-choline-containing lipids (either monomeric or micellar) rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Therefore, two functional sites on the enzyme have to be assumed an activator site and a catalytic site (6). 

Phospholipases (PLs) are a ubiquitous group of enzymes that share the property of hydrolyzing a common substrate, phospholipid. Nearly all share another property they are more active on aggregated substrate above the phospholipid s critical micellar concentration (cmc). As shown in Fig. 1, phospholipases have very low activity on monomeric substrate but become activated when the substrate concentration exceeds the cmc. The properties of phospholipids that define the aggregation state (micelle, bilayer vesicle, hexagonal array, etc.) are described in Chapter 1. 

Micelles, however, differ in important aspects from biological membranes. Micelles have typical diameters of 5 nm and therefore may be too small to mimic organellic membranes. In order to understand the conformational changes that occur upon membrane binding of monomeric ASYN, SDSL EPR was performed with ASYN bound to phospholipid vesicles, e.g., small or large unilamellar vesicles (SUVs or LUVs, respectively). 

A Protein Example Phoshpholipase A2 Pancreatic phospholipase A2 is an enzyme of molecular weight 14,000, which catalyses the hydrolysis of 2-acyl ester bonds in a variety of naturally occurring phospholipids. The enzyme is secreted as a zymogen, which is activated by tryptic cleavage of the N-terminal heptapeptide. Both the enzyme and its precursor show catalytic activity towards monomeric lipids, but in contrast to the precursor the active enzyme shows a tremendous rate increase when it acts upon organized lipid structures such as micelles and bilayers (23). 

This chapter limits itself to P-NMR studies on phospholipids and lysophospholipids in monomeric and micellar states and focuses on the identification of species and aggregation states, as well as on the dynamic processes of migration and reaction kinetics. For this purpose, micelles and mixed micelles are defined as dilute isotropic solutions of phospholipids, either with or without detergents that form spontaneously and are at thermodynamic equilibrium diis definition specifically excludes sonicated or small unilamellar vesicles as well as membranes. These are covered by Smith and Ekiel (Chapter 15). This limitation in scope necessarily requires heavy reliance on work from the laboratory of the authors of this chapter. 

Phospholipids can be synthesized with short fatty acid chains to make them water soluble, and the physical properties of such phospholipids have been studied extensively by Tausk et al. (1974a,b,c). Their critical micelle concentration (CMC) depends on chain length and has been determined by a variety of methods for a number of PC derivatives. It was found that the 3 P-NMR signal is chemically shifted in going from the monomeric form to the micellar form of dihexanoyl PC (Roberts et ai, 1979). This difference can be used (Pluckthun and Dennis, 1981) to determine the CMC according to... 

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