Reaction with phosphatidic acid

Figure 2.13. Histamine H,-receptor-mediated inositol phospholipid hydrolysis. Stimulation of H,-receptors leads to activation of a phospholipase C. probably via a guanine-nucleotide regulatory protein (N). which catalyses the hydrolysis of phosphatidylinositol 4.5 -bisphosphate (PIP2) to give inositol trisphosphate (IP3) and 1,2-diacylglycerol (DG). IP3 is then broken down by phosphatases to eventually yield free myo-inositol. Lithium ions can inhibit the conversion of inositol 1-phosphate (IP,) to myo-inositol. Free inositol then interacts with CDP-diacylglycerol,formed by a reaction between phosphatidic acid (PA) and CTP, to yield phosphatidylinositol (PI). Phosphorylation of PI by kinases completes the lipid cycle by reforming PIP2. Modified from [147,148].

The enzymes responsible are found in the albatross salt gland (Hokin and Hokin, 1960), erythrocyte membranes (Holdn and Hokin, 1961), and brain microsomes (Hokin and Hokin, 1959b). Mcllwain (1963) has criticized the scheme on quantitative grounds, making the point among others that the observed rates for the enzymatic reactions involved in cerebral tissue are about 0.02% of tiie rates of cation transport. It is still not proved, in fact, whether the increased exchange of phosphate with phosphatidic acid in membranous systems in the presence of acetylcholine is a secondary result of an action of the drug on some other system. 

On the basis of such studies, Hokin proposed a cyclic type of reaction in which sodium combines with phosphatidic acid to yield sodium phosphatidate. Sodium phosphatidate is assumed to be formed at the inner side of the membrane. 

Recent work in the general field of lipid biochemistry has revealed that although the carbon skeleton of serine is extensively used in the biosynthesis of ethanolamine (and hence choline and acetylcholine), decarboxylation of free serine does not occurnor is the ethanolamine formed from serine found free. It appears that the hydroxyl group of serine is first esterified with phosphatidic acid by reaction with cytidine diphosphate diglyceride (Figure 21). Phosphatidyl... 

The valency of the metal ion changes in every step so that a single atom of heavy metal (Me) may produce many free radicals. Metal chelating compounds, such as citric, tartaric or phosphoric acids, ascorbic acid, phytin or phosphatidic acids, combine with metals to form non-reactive compounds so that the oxidation reactions are inhibited and natural food antioxidants are saved. 

It can be seen from Figure 1 that the choline-containing phospholipids, phosphatidylcholine and sphingomyelin are localized predominantly in the outer monolayer of the plasma membrane. The aminophospholipids, conprising phosphatidylethanolamine and phosphatidylserine, by contrast, are enriched in the cytoplasmic leaflet of the membrane (Bretcher, 1972b Rothman and Lenard, 1977 Op den Kamp, 1979). The transmembrane distribution of the minor membrane lipid components has been determined by reaction with lipid-specific antibodies (Gascard et al, 1991) and lipid hydrolases (Biitikofer et al, 1990). Such studies have shown that phosphatidic acid, phosphatidylinositol and phosphatidylinositol-4,5-fc -phosphate all resemble phosphatidylethanolamine in that about 80% of the phospholipids are localized in the cytoplasmic leaflet of the membrane. 

The initial reactions produce phosphatidate, in which the two hydroxyl groups of glycerol 3-phosphate are esterified with long-chain fatty acids, catalysed by enzymes known as acyltransferases. An important point is that, due to the difference in specificities of the acyltransferase enzymes. 

Triacylglycerols are formed by reaction of two molecules of fatty acyl-CoA with glycerol 3-phosphate to form phosphatidic acid this product is dephosphoiylated to a diacylglycerol, then acylated by a third molecule of fatty acyl-CoA to yield a triacylglycerol. 

Synthesis of most phospholipids starts from glycerol-3-phosphate, which is formed in one step from the central metabolic pathways, and acyl-CoA, which arises in one step from activation of a fatty acid. In two acylation steps the key compound phosphatidic acid is formed. This can be converted to many other lipid compounds as well as CDP-diacylglycerol, which is a key branchpoint intermediate that can be converted to other lipids. Distinct routes to phosphatidylethanolamine and phosphatidylcholine are found in prokaryotes and eukaryotes. The pathway found in eukaryotes starts with transport across the plasma membrane of ethanolamine and/or choline. The modified derivatives of these compounds are directly condensed with diacylglycerol to form the corresponding membrane lipids. Modification of the head-groups or tail-groups on preformed lipids is a common reaction. For example, the ethanolamine of the head-group in phosphatidylethanolamine can be replaced in one step by serine or modified in 3 steps to choline. 

Acetylcholineesterase Bilayer lipid membranes were prepared by adding a solution of egg phosphatidylcholine and dipalmi-toyl phosphatidic acid dropwise into the surface of aqueous 0.1 M KC1/10 mM HEPES, near the Saran Wrap partition of a two compartment plexiglass cell. A portion of AChE solution in 10 mM Tris hydrochloride buffer solution of pH 7.4 was applied. The electrolyte level was momentarily dropped below the orifice and raised to form a membrane. The membranes were used as transducers for the reaction of AChE with ACh. An external voltage (25 mV) was applied across the membrane between two Ag/AgCl reference electrodes. Enzymatically generated hydronium ion causes transient current due to alteration of the electrostatic field by the ionization of dipalmitoyl phosphatidic acid. The response delay time was directly related to the substrate concentration where acetylcholine can be determined from 1 pM upto mM level. [113]... 

In the work-up of the diethyl ether reaction, the solvent is removed (in a hood) under a stream of nitrogen. Then the ether-free aqueous dispersion is mixed with 10 ml of a 5 mM EDTA solution to bind the calcium ions. Without the latter treatment, the calcium salt of the phosphatidic acid cannot be recovered (easily) from the reaction mixture. Thus, subsequent to the EDTA treatment, the sample is mixed with sufficient chloroform and methanol to make a mixture of chloroform-methanol-water (1 2 0.8, v/v). Then 0.5 volume chloroform and 0.5 volume water are added, and the mixture is vigorously shaken for 1 min and then allowed to separate into two phases. The upper, water-rich phase contains free nitrogen base—in this case, choline. It can be analyzed by the techniques described earlier. 

In experiments with PAF as the agonist, this reaction would show the level of PIP2 to increase nearly 135% compared to a control within 1 min and would also show the PIP level to increase to approximately 225% of the control in 3 min. Changes in the PI level were not significant. Of importance, phosphatidic acid levels rose very rapidly within 30 sec to a value approximately eightfold greater than that of the control. 

The synthesis of triacylglycerol takes place in the endoplasmic reticulum (ER). In liver and adipose tissue, fatty adds in the cytosol obtained from the diet or from de novo synthesis of palmitic add become inserted into the ER membrane. The reactions are shown in Fig. 13-10. Membrane-bound acyl-CoA synthetase activates two fatty acids, and membrane-bound acyl-CoA transferase esterifies them with glycerol 3-phosphate, to form phosphatidic acid. Phosphatidic acid phosphatase releases phosphate, and in the membrane, 1,2-diacylglycerol is esterified with a third molecule of fatty acid. 

The product is phosphatidic acid, itself an important substance (see page 31) and the starting point for the phosphatides and related lipids. The conversion of phosphatidic acid into triglyceride demands first the removal of the phosphate through hydrolysis by the enzyme phosphatidic acid phosphatase (an irreversible reaction). The product is diglyceride, which can react with a final fatty acyl-CoA to yield triglyceride (14,15) ... 

Abrahamson et at (34) observed a similar departure from the latter sequence of calcium and magnesium in their reaction with a phosphatidic acid dispersion at pH 7. Shah and Schulman (35) also found a higher tendency of Ca2+ than Mg2+ to interact with a monolayer of dicetyl phosphate at pH 5.6 as judged by surface pressure measurements although surface potential measurements indicated little difference between the two ions as did potential measurements on phosphatidyl serine mem-... 

CTP phosphatidate cytidylyltransferase has been observed in plastids, mitochondria, and ER. In all three compartments, the CDP-diacylglycerol derived from phosphatidic acid is used in the synthesis of phosphatidylglycerol in mitochondria, the reaction of phosphatidylglycerol with a second CDP-diacylglycerol then produces cardiolipin. The ER can also incorporate CDP-diacylglycerol into phosphatidylinositol and phosphatidylserine. 

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