Phosphatidic acids, isolation

The action of the complex is compared to that of the acidic PL phosphatidyl serine, phosphatidyl inositol and phosphatidic acid. These PL first remove calcium, and a small amount of phosphate from the metastable solution, forming a material similar to the complex isolated from bone, and then form HA. The rate of HA proliferation, once phosphatidyl serine and phosphatidyl inositol are converted to Ca-PL-P04 complexes, is the same as the rate observed for comparable weights of the complex isolated from bone110 ... 

The lower, chloroform-rich phase is separated carefully from the protein-containing interface, and then it is washed twice with methanol-water (10 9, v/v) and the washes are discarded. The chloroform layer contains the phosphatidic acid (as a sodium salt) and can be isolated by acetone precipitation. The yields can be of the order of 90-95%. One alternative route to identification of the chloroform-soluble material is to analyze it for total phosphorus and total fatty acid ester (see procedures described earlier). In the case of diacylphosphatidylcholine as the substrate, the fatty acid ester/P molar ratio should be 2.0. Another approach is to subject the chloroform-soluble fraction to preparative thin-layer chromatography on silica gel H (calcium ion free) in a two-dimensional system with a solvent system of chloroform-methanol-28% ammonium hydroxide (65 35 6, v/v) in the first direction and a solvent system of chloroform-acetone-methanol-glacial acetic acid-water (4.5 2 1 1.3 0.5, v/v) in the second direction. The phosphatidic acid will not migrate far in the basic solvent Rf 0.10) and will show an Rf value one-half of that of any remaining starting substrate (fyO.40) in the second solvent. Of course with a simple substrate system, one can use the basic solvent in one dimension only... 

Glycerophosphatides. These lipids are mainly acyl derivatives of a-glycerophosphoric acid and often are called phospholipids. The simplest glycerophosphatides are the phosphatidic acids, which contain a-glycerophosphoric acid esterified with two fatty acids (Fig. III-37). Small quantities of phosphatidic acids have been isolated from a wide variety of plant and animal tissues. It is doubtful that these compounds exist in large amounts in tissues, because more complex glycerophosphatides are readily hydrolyzed by enzymes that are widely distributed in such tissues, yielding phosphatidic acids. Phosphatidic acid is a crucial intermediate in the biosynthesis of phospholipids. 

The linkage of the mj/o-inositol to the phosphatidic acid has not yet been determined with certainty the inositolphosphoric acid, isolated after hydrolysis of the intact phosphatide (or of the water-soluble portion, obtained by alkaline hydrolysis), gives, on paper chromatography, a mixture of inositol 1(3)-phosphate and a small proportion of inositol 2-phosphate (separated on paper by the method of Pizer and Ballou ). This indicates that, in the phospholipid, the m /o-inositol is probably linked by its C-1- or C-3-hydroxyl group to phosphoric acid. 

Although phosphohpids like acetal phosphatidic acid and lysolecithin have undoubted pharmacological activity, it is the opinion of this reviewer that, until they can be isolated by very mild conditions, their existence in the tissues examined must remain an open question. To say this is not to deny a lipid pharmacology, since prostaglandins in particular are lipids, but to reserve judgment on phospholipid pharmacology on present evidence. 

There is a scarcity of data for the analyses of glyoxysomal membranes and plasma membranes of plant cells. The glyoxysomal membrane from castor bean (Ricinus communis L.) endosperm has PC as the major lipid together with PE, 80% of the phospholipid can be accounted for. Plasma membranes isolated from oat Avena saliva L.) roots showed relatively lai ge amounts of phosphatidic acid (PA), (1,2-diacyl-jn-glycerol 3-phosphate) and an unknown lipid (Keenan et al., 1973). It is possible that some lipid degradation took place in the preparation of this sample. Further analyses of the lipid composition would be most appropriate. The analyses of Keenan et al. (1973) indicated that only 28.6% of the plasma membrane lipid was phospho-... 

Also, isolated mitochondria were found only capable of synthesizing acidic phospholipids, such as phosphatidic acid, phosphatidylgjycerol, di-phosphatidylglycerol, and phosphatidylinositol, despite the fact that the main phospholipids of mitochondrial membranes are phosphatidylcholine and phosphatidylethanolamine (Douce, 1%8 Sumida and Mudd, 1968 Bahl etal., 1970). 

Fig. 10. Mechanism explaining the acylation of -glycerol 3-phosphate in the envelope membranes. El, Acyl-CoA synthetase E2, acyl-CoA in-glycerol 3-phosphate acyltransferase E3, acyl-CoA acyl-sn-glycerol 3-phosphate acyltransferase E4, phosphatidic acid phosphatase. El, E3, and E4 are firmly bound to the chloroplast envelope. E2 is probably loosely bound to the envelope and is released into the medium during the isolation (see Fig. 12) (reproduced from Douce and Joyard, 1979a, by permission).

Like glycolipids, phospholipids are also a elass of polar lipids. Data in Table 3.2 show that phospholipids in natural rubber are normally below 0.6% w/w. A recent study showed that the major components were phosphatidyl choline (PC) and lysophosphatidyl choline (LPC), as shown in Table 3.5. Other minor components were phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), lysophosphatidyl inositol (LPI), and metal phosphatidates (MP) or phosphatidic acid (PA). Unlike neutral lipids and glycolipids, the acyl components of phospholipids normally contain very low levels of furanoic acid, except for certain rubber clones. The isolated fatty acids are mainly palmitic, stearic, oleic and linoleic acids. ... 

The major action of acetylcholine is at post-synaptic membranes, so it is surprising that this transmitter affects phospholipid metabolism in isolated synaptosomes. As Fig. 2 indicates, a fragment of post-synaptic membrane is usually attached to synaptosomes, but this will not have access to the metabolic pathways involved in phospholipid synthesis. Transmission of the nerve impulse involves phospholipid changes in both pre-and post-synaptic membranes. L. E. Hokin (1969) concluded from pre-ganglionic denervation experiments with sympathetic ganglia that there was a pre-synaptic phosphatidic acid effect but that the phosphatidylinositol changes induced by acetylcholine were largely post-synaptic. 

Breakdown and resynthesis of phosphatidylinositol and phosphatidic acid are closely associated with the events following depolarization of isolated synaptosomes. The most important of these events is transmitter release and the weight of the present evidence favours the view that exocytosis is the mechanism of release. This is supported by our finding that major phospholipid effects are seen in the synaptic vesicle fraction. Work with pre-labelled synaptosomes showed that stimulation led to loss of phosphatidylinositol from this fraction. The enzyme converting it to diacylglyce rol is most likely to be involved. Entry of calcium... 

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