Lipid-Water Complexes

The author and coworkers investigated the interfacial phenomena happening at the interface between a biological membrane and physiological solution. A biological membrane consists of various molecules such as lipids, proteins, and cholesterols. The physiological solution mainly consists of water molecules but contains various ions and small organic molecules. At the interface between these two domains, they interact with each other to 

Biological membranes are so complicated that a lipid membrane is often used as a model sample for their studies. The author and coworkers investigated the interface between a DPPC bilayer and PBS buffer solution. At the lipid-water interface, water molecules interact with lipid head groups to show nonuniform distribution. Since an AFM tip interacts with water molecules as well as the lipid head groups, the force distribution detected by AFM should be influenced by the water distribution as well as by the surface corrugation. 

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As depleted in Scheme A, valinomycln forms a lipid-soluble complex with K an transports it across the membrane. An increase in internal K will be accompanied by the movement (diffusion) of the Cl counter ion, in some as yet unknown manner, to maintain electroneutrality. The increase in the matrix concentration of KCl will result in the osmotic Influx of water. Swelling is measured as a decrease in absorbance. The extent of swelling under control conditions is shown in the 0 trace. Quercetin inhibited, in a concentration-dependent manner, the extent of osmotic swelling. 

The major polymers that make up the wall are polysaccharides and lignin. These occur together with more minor but very important constituents such as protein and lipid. Water constitutes a major and very important material of young, primary walls (2). The lignin is transported in the form of its building units (these may be present as glucosides) and is polymerized within the wall. Those polysaccharides which make up the matrix of the wall (hemicelluloses and pectin material) are polymerized in the endomembrane system and are secreted in a preformed condition to the outside of the cell. Further modifications of the polysaccharides (such as acetylation) may occur within the wall after deposition. Cellulose is polymerized at the cell surface by a complex enzyme system transported to the plasma membrane (3). 

Following extraction, an efficient way of initiating the isolation of carotenoids is to saponify the extract. This removes many of the unwanted lipids present in the sample as well as chlorophyll. The saponification by-products, which to a great extent are sodium or potassium salts, are easily separated by an aqueous solution of a highly polar salt. The addition of water also helps wash off excess alkali and other water-soluble and water-complexed compounds. This procedure hydrolyzes xantho-phyll esters to form the hydroxylated carotenoid. 

Prior to phospholipid analysis, it is imperative to extract the lipids from their matrix and free them of any nonlipid contaminants. Phospholipids are generally contained within the lipid fraction, which may be recovered by the traditional Bligh and Dyer or Folch extraction procedure (9,22). In any phospholipid extraction method it is recommended to include a rather polar solvent in addition to a solvent with high solubility for lipids. The former is needed to break down lipid-protein complexes that prevent the extraction of the lipids in the organic phase. Traditionally, mixtures of chloroform and methanol (especially 2 1, v/v) have been recommended. These are washed with water or aqueous saline to remove nonlipid contaminants. Comparing the recovery of phospholipids, Shaikh found that the neutral phospholipids PC, PE, SPH as well as DPG were nearly quantitatively extracted by all solvent systems studied (Table 1), although Bligh and Dyer, in which the lower phase was removed only once, was somewhat worse (23). 

The present knowledge about molecular organization in lyotropic liquid crystalline phases is summarized. Particular attention is given to biologicaly relevant structures in lipid-water systems and to lipid-protein interactions. "New findings are presented on stable phases (gel type) that have ordered lipid layers and high water content. Furthermore, electrical properties of various lipid structures are discussed. A simple model of l/l noise in nerve membranes is presented as an example of interaction between structural and electrical properties of lipids and lipidr-protein complexes. 

The biochemical work described in Chapter 5 indicated that the microbubble surfactant mixture actually represents a glycopeptide-lipid-oligosaccharide complex, which is reversibly held together by both hydrogen bonding and nonpolar interactions. The experiments described below were undertaken to examine in detail the surface properties of the microbubble surfactant complex at the air/water interface (ref. 361). 

Bile salts are substances derived from sterols, which make up a substantial part of the solid matter in bile and which play a central role in lipid absorption, by virtue of their surface-active properties. The structure and properties of these salts have been reviewed by Haslewood (305) and Heaton (316). Bile salts essentially have molecules of detergent type hydrocarbon, with a fat-dissolving part and a polar, water-attracting part. The fat-dissolving part consists of the bulk of the steroid nucleus. The hydroxyl groups are so distributed that hydration can readily take place the remainder of the molecule will dissolve the fatty phase. Emulsification of fat/water complexes can thus occur easily. The terms bile acid and bile salt are used somewhat interchangeably in the literature. 

While there is no doubt that the major attractive forces maintaining lipids in membranes are noncovalent in nature, there is excellent evidence in the literature showing that a small percentage of the membrane lipids is covalently linked to membrane proteins. These lipids are highly specific in nature and will be discussed briefly below. Normally these lipid protein complexes are not found in the organic solvent phase of a typical (lipid) extraction procedure. Rather they would be found in the water-rich phase of such an extraction approach. Basically there are four specific classes of lipid covalent binding to protein ... 

It is also important to remember that wheat gluten and dough are complex materials, consisting not only of protein and water, but also starch-, lipid-, water- and salt-soluble proteins and smaller carbohydrates, and so on. The properties of these materials and their interactions with the gluten proteins are poorly understood but can be expected to also influence the viscoelastic properties. The challenge therefore is to understand gluten structure at the molecular level and how this structure interacts... 

There were two new aspects. One was the fact that this molecule is an integral membrane protein. Structures of such molecules had not been determined before. They sit in a membrane, which is a lipid bilayer about 50 angstroms thick. This lipid bilayer is a very different environment for a protein than water is. Many of the proteins we know are water-soluble so they present a polar surface to their environment. Membrane proteins have two types of surface they present a hydrophobic surface in that part, which is inside the membrane, and a polar surface in the part that sticks out. That makes them very different to handle, to purify, to crystallize from water-soluble proteins. When we crystallize it, we have to crystallize the whole protein, both parts inside and outside of the membrane. We tried to create conditions in which both the hydrophobic surfaces and the hydrophilic surfaces of the protein were in the correct environment. That was done by coating the hydrophobic surface with so-called detergent micelles. These are molecules that have hydrophobic and hydrophilic ends. They are. similar to lipids but they don t form planar bilayers. They tend to form spherical micelles. Under the right circumstances these micelles can form a belt around the hydrophobic surface of the protein, and thus replace the lipid. This complex of the protein and the detergent has many properties like a water-soluble protein and can be crystallized as water-soluble proteins. So this was a new feature. 

Cubic lipid phases have a very much more complex architecture than lamellar and hexagonal phases. Their structural characteristics have been elucidated only very recently, and it has become clear that their subtleties are the key to a variety of biological problems. We will consider those subtleties in some detail. The three fundamental cubic minimal surfaces - the P-surface, the D-surface and the gyroid (or G-surface), introduced in Chapter 1, can all be foimd in cubic lipid-water phases. The lipid bilayer is centred on the surface with the polar heads pointing outwards. Water fills the labyrinth systems on each side of the surface. These cubic phases will be termed Cp, CD and CG/ respectively. It is likely that there are other more complex IPMS morphologies in cubic phases of lipid-water mixtures, as yet uncharacterised. 

One important experimental result was available, the quantitative measurement of the fraction of each secondary structural element by circular dichroism (CD) on purified lipid-protein complexes. This provided a constraint that allowed a careful evaluation of the secondary structure predictions derived from the various approaches, some of which were developed for water-soluble proteins and therefore of uncertain reliability for proteins in a lipid environment. The data from these analyses were combined using an integrated prediction method to arrive at a consensus secondary structure model for each protein. The integrated method involved 36 steps, with independent predictions at each step. The final model was based on an evaluation of the various predictions, with judicious intervention by the authors. As an aid to developing the appropriate weighting of all the data, they carried out the analysis for apoE-3 without reference to the available crystal structure (Wilson et al., 1991), then used the known structure of the HDL-binding amino-terminal domain of apoE-3 as feedback to reevaluate the weighting. 

Fig. 9.3 Kinetic model illustrating the covalent inhibition of a lipolytic enzyme at a lipid/ water interface. Symbols and abbreviations are as follows A, total interfacial area (surface) V total volume (volume) E, free enzyme concentration (molecule/volume) , interfacial enzyme concentration (molecule/ surface) S, interfacial concentration of substrate (molecule/surface) I, interfacial concentration of inhibitor (molecule/surface) P, product concentration (molecule/volume) E S, interfacial enzyme-substrate complex... 

On the basis of the above mentioned kinetic and mass spectrometry analysis, a model was proposed for the covalent inhibition of HPL by orlistat in the aqueous phase as well as its partial reactivation at the lipid-water interface (Fig. 9.16). This model takes into account the putative existence of two different forms of the covalent orlistat/lipase complex. 

Fig. 9.16 Kinetic model illustrating the inhibition of HPL by orlistat in the aqueous phase and its reactivation at a lipid-water interface. The following symbols and abbreviations are used here E, free enzyme (molecule/volume) E, interfacial enzyme (molecule/surface) FA, fatty acid at the interface (molecule/surface) E -FA, interfacial enzyme-fatty acid complex (molecule/surface) THLc, closed reactive orlistat in the bulk (molecule/volume) THLo, open non-reactive orlistat at the interface (molecule/surface) -THLO, form 1 of cova-...

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