Phospholipids analysis, inositol

These treatments convert to ionic substances, and remove, nearly all constituents of natural materials the acid treatments release any inositol present as phosphate, or combined in phospholipids, glycosides, etc. Glycerol remains in the deionized sample, but it can be oxidized separately, or be removed by heat decomposition or by repeatedly evaporating the solution to dryness. Such polyhydric alcohols of greater chain length as erythritol and mannitol, when present, would still interfere. However, corrections can be made for these compounds by determining the formaldehyde which they form on periodate oxidation, or they may be removed by chromatography on filter paper. The micro-periodate method is well suited to the analysis of samples eluted from filter paper, provided that care is exercised to remove the tiny particles of cellulose which are usually found in such eluates. 

Subsequent to recovery of the total lipids of a cellular preparation as a chloroform-soluble fraction, the total phosphorus content can be determined (see Chapter 3) and then, depending on the amount of lipid phosphorus (or whether the preparation is radiolabeled or not, see below), analytical and/or preparative thin-layer chromatography can be undertaken. In either case, if the experimental protocol is centered on a signal-transduction process, then there may be insufficient material for a phosphorus analysis. In the latter instance, the cellular preparation is prelabeled with 32P or [3H]inositol and the labeled products are located by autoradiography. A preferred type of adsorbent (for thin-layer chromatography) is Merck silica gel 60 (oxalate impregnated). An effective solvent for separation of the phosphatidylinosi-tols and other lipids is chloroform-acetone-methanol-acetic acid-water (80 30 26 24 14, v/v). The approximate / values of cellular phospholipids under these conditions are presented as follows ... 

L. E. Bertello, M. F. Goncalvez, W. Colli, and R. M. de Lederkremer, Structural analysis of inositol phospholipids from Trypanosoma cruzi epimastigote forms, Biochem. J., 310 (1995) 255—261. 

Similarly to T. cruzi, inositol phospholipids have been identified in T. brucei brucei (44). Moreover, two distinct PI synthase activities (CDP-diglyceride-dependent and -independent) are present in membrane fractions. In the absence of added lipid precursor, the incorporation of free inositol into PI is strongly stimulated by the addition of Mn " and less by Mg . Addition of exogenous CDP-diglyceride to the system increases the uptake of inositol by a factor of 1.5 to 3.5. These inositol phosphates may mediate part of the T. brucei brucei response to external signals. Previous work have shown that transformation of bloodstream forms to procyclic cells can be induced by cis-aconitate or citrate. Analysis of inositol phosphates in [ H]inositol-labeled cells exposed to ds-aconitate revealed that levels of InsP3 increased rapidly (maximum levels at one hour) and subsequently returned to basal level. The levels of InsP and InsP2 were not or little affected by cis-aconitate. 

The ability of PI synthetase to use 5-deoxy-5-fluoro-myo-inositol (4) as a substrate was confirmed by use of a radiolabeled compounds as shown in Figure 7. PI synthetase incorporated the analog into lipid in a time-dependent manner. The incorporation was absolutely dependent on the presence of CDP-diglyceride and was inhibited by the presence of myo-inositol (1) in the incubation mixture, as expected for PI synthetase. Chromatography of the reaction mixture revealed that a single radiolabeled product was formed with a mobility similar to, but distinct from, that of PI. Subsequent analysis has shown that the product is converted to a water-soluble form on mild alkaline hydrolysis and yields 5-deoxy-5-fluoro-myo-inositol (4) on treatment with phospholipase D, in agreement with the formation of phosphatidyl-5-deoxy-5-fluoro-myo-inositol as the product (data not shown). Determination of the absolute structure of these phospholipids awaits large-scale enzymatic synthesis, isolation of the product, and studies by mass spectrometry and NMR spectroscopy. 

Figure 9.12 Direct electrospray ionization-mass spectrometry analysis of human erythrocyte plasma membrane phospholipids (A) A positive-ion electrospray ionization (ESI) mass spectrum of erythrocyte plasma membrane phospholipid extract showing 14 molecular species of glycerophospholipids and 4 molecular species of sphingomyelin (B) A negative-ion ESI mass spectrum of the same extract of plasma membrane phospholipids showing more than 25 molecular species of ethanolamine glycerophospholipids and 8 molecular species of serine and inositol glycerophospholipids. Reprinted with permission from Han, X. and Gross, R. W., Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids, Proc. Natl Acad. Scl USA, 91(22), 10635-9. Copyright (1994) National Academy of Sciences, USA.

The chemistry of ROS has been studied by several laboratories, and recently reviewed by Daemen (1973). Over 90% of the protein is rhodopsin, a photosensitive glycoprotein of molecular weight around 35,000, which is imbedded in a lipid bilayer. Phospholipids make up about 96% of the lipids of cattle ROS and cholesterol is the major component of the neutral lipid fraction. Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) are the major phospholipids in all species examined, with phosphatidyl serine (PS), phosphatidyl inositol (PI), and sphingomyelin (SPh) present in lesser amounts (Anderson and Maude, 1972). Detailed analysis of the photoreceptor membranes of vertebrate species ranging from frogs to humans have revealed a fairly constant phospholipid class and protein composition (Basinger and Anderson, unpublished). 

Methods for the quantitative chromatographic analysis of inositol phosphates and inositol phospholipids have been reviewed. ... 

Bacterial membranes. The cytoplasmic membrane of bacteria has both usual and unusual features. When the cell wall is completely hydrolysed by lysozyme, this membrane becomes the outer layer. It is 60-100 A thick and sometimes extends into the cytoplasm as a few simple protrusions (Mitchell and Moyle, 1956b Hughes, 1962). It forms about 10 per cent of the dry weight of the cell and has a lipid content of about 25 per cent. There is usually little lipid elsewhere in the cell. An analysis of the lipid of M. lysodeikticus shows that 80 per cent is phospholipid, which is mainly diphosphatidyl glycerol, but some phosphatidyl inositol is also present. 

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