Phospholipids chemical characterization

Chemical characterization hypothesizes that certain molecules are unique and representative of individual microorganisms, and therefore the mass spectra can be characteristic of given species. A typical bacterial spectrum obtained by MALDI-TOF usually contains between 20 and 40 large peaks, mainly produced by proteins, phospholipids, and cyclic lipopeptides, in the MW range of 4—20 kDa.31 ESI is less frequently used samples containing intact cells can clog electrospray devices, and the formation of multiply charged species complicates the spectra. 

Wohnsland and Faller ([175] performed measurements using a thin (9-10 //in) supported, phospholipid-free hexadecane layer. To validate their model, they used 32 well-characterized chemically diverse compounds. The permeability values obtained with their model could be correlated with known human absorption values if the maximum permeability obtained at different pH was taken into account. However, several disadvantages are related to this method. For hydrophilic drugs, hexadecane by itself has an increased barrier function in comparison with membranes. In addition, the hexadecane layers are not very stable, which makes this assay difficult to apply as a routine screening method. The advantage of this PAMPA setup is that it appears to be a satisfactory substitute for obtaining alkane-water partition coefficients, which are usually very difficult to measure directly, due to the poor solubility of drug molecules in alkanes. 

As with other multisubunit enzymes (e.g., allosteric enzymes), the structural integrity of a membrane-bound enzyme primarily is maintained by noncovalent interactions such as hydrogen bonding, electrostatics, and hydrophobic interactions. Hydrophobic polypeptides (or hydrophobic portions of polypeptides) apparently are used to anchor the enzymes to the membrane through interactions with phospholipids. Therefore, I would characterize the interaction between the enzyme and membrane as chemical in nature rather than as geometric. ... 

As a first approach, one must undertake to isolate these substances from a cell, purify them to apparent homogeneity, and then determine their chemical structure. Only with such an approach can one hope to interpret their exact role in cellular reactions. The next chapter will center on the isolation, purification, and initial characterization of cellular phospholipids. 

In the direct chemical attack for proof of structure of phosphatidylcholine (see the section entitled Glycerophosphochline Characterization ) the decision was made to subject this phospholipid to a base-catalyzed methanolysis. As noted, two products are formed, namely, glycerophosphocholine and the methyl esters of the long-chain fatty acid substituents on the intact phosphatidylcholine. Since the analytical approach to proof of structure of the glycerophosphocholine has been achieved, it is logical now to consider the other product, the methyl esters. 

The ligand binding pocket of USP is filled by a fortuitous phospholipid co-purified and co-crystallized with the USP LBD that was fiirther characterized by mass-spectroscopic and chemical analysis [57]. In a similar way, recent crystallographic investigations of the retinoid-acid related orphan receptor (3 (ROR (3) [58] and of the heterodimeric complex RARa/RXRa [30] revealed an E.coli endogeneous fatty acid in the ROR (3 and in the RXRa subunit, respectively. 

In practice, commercial lecithin products are not marketed by phospholipid content, but rather by a set of unique chemical and physical properties. These properties, as indicated by product specihcations, must be understood because they are used to characterize specihc lecithin types. 

Black (1968) reviewed briefly the present status of our knowledge of the chemical nature of the organic phosphate of soils and concluded that if the identified organic phosphorus is summed, one has about 2% of the total present in nucleic acids, 1% in phospholipids, and 35% in inositol phosphates, making a total of 38% accounted for. The nature of the remainder is unknown. (Also see Cosgrove, 1967.) Much further research, initially on methods, will be needed before the organic phosphorus can be characterized with any satisfactory degree of exactness. 

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