Phosphate, inorganic Phosphatides

The path of conversion of inorganic phosphate to phosphatide is not known. Glycerophosphate, diglycerides, neutral fat, choline phosphate, phosphoproteins, or other compounds may be involved as intermediates. That aminoethylphosphoric acid can be excluded from this group of substances follows from the work of Chargaff and Keston (38). Experiments in which 80 mg. of labeled disodium aminoethyl phosphate was administered to adult rats by subcutaneous injection showed that the body was unable to utilize aminoethylphosphoric acid as such for the synthesis of cephalin. Of the administered as aminoethylphosphoric acid, 28% was found to be excreted through the kidneys in the course of eight hours in these experiments. 

C. In the liver, 2 fatty acyl CoAs react with glycerol 3-phosphate to form phosphatidic acid, which releases inorganic phosphate to form a diacylglycerol. The diacylglycerol reacts with fatty acyl CoA to form a triacylglycerol. 

As mentioned above, there is evidence that the initial phospholipid change on excitation is hydrolysis of phosphatidylinositol (Fig. 1). Hydrolysis of phosphatidate to diacylglycerol has also been postulated to occur in synaptosomes incubated with acetylcholine (Schacht Agranoff, 1974). These hydrolytic changes would not be detected in experiments measuring incorporation of labelled inorganic phosphate into synaptosomal phospholipids. We have therefore labelled synaptosomal phospholipids with 32p vivo and studied the loss of label caused by electrical stimulation. 

Figure 2. The so-called canonical phosphoinositide pathway . The continuous phosphorylation/dephosphorylation reactions allow a steady-state level of Ptdins, PtdIns(4)P and PtdIns(4,5)P2 in the plasma membrane (PM). Cleavage of PtdIns(4,5)P2 by phospholipase C (PLC) generates the two well-known second messengers, inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and diacylglycerol (DAG). Besides its role as a protein kinase C (PKC) activator, DAG can be phosphorylated to phosphatidic acid (PA). The resynthesis of Ptdins from inositol and PA occurs mainly in the endoplasmic reticulum (ER). PPi, inorganic phosphate. PA-Pase, phosphatidic acid phosphatase. PA-TP, phosphatidic acid transport protein. PtdIns-TP, phosphatidylinositol transport protein. CDP-DAG, cytidine diphosphate-diacylglycerol. CMP, CDP and CTP, cytidine mono-, di- and triphosphate, respectively.

The reaction is freely reversible and the enzyme catalyzing it has been called DPN pyrophos-phorylase. This transfer of one 5 -nucleotide to another produces a pyrophosphate bond between them. The reaction is the prototype of a large number of such nucleotidyl transfers to other phosphate compounds. These include transfers to various sugar phosphates to form the nucleoside diphosphate sugar coenzymes, to choline phosphate to form C3fiidine diphosphate choline, and to phosphatidic acid to form cytidine diphosphate diglyceride. This nucleotidyl transfer is the protot)q)e also for transfers of nucleotides to produce mixed acid anhydrides with fatty acids, amino acids, and sulfates. In each instance inorganic pyrophosphate is produced this is also true of the nucleotidyl transfers which produce RNA and DNA. 

If we make the assumption that phosphate ions or phosphorus-containing precursors which attain rapid equilibrium with the phosphate ions are incorporated into the phosphatide molecule, we can determine the rate of renewal of phosphatide molecules by comparing the specific activity of the phosphatide phosphorus at the end of the experiment with the mean specific activity of the intracellular inorganic phosphorus prevailing during the experiment. This calculation involves the further assumption that it is the intracellular, inorganic phosphorus which is incorporated into the phosphatide molecule. The rate of renewal of phosphatides extracted... 

That the rate of renewal of phosphatides in liver is accelerated if the fat content of the circulation is increased was also shown in experiments on perfused cat liver (68). With normal blood, 1.5% of liver phosphatide phosphorus was found to be replaced by active inorganic phosphorus added to the blood as sodium phosphate in the course of 2.5 hours, while with lipemic blood 2.7% was renewed. Thus, the effect of ingested fat on the rate of renewal of phosphatides is very pronounced in those organs which, like the intestinal mucosa and the liver, play a predominant part in fat metabolism. 

The question whether a tissue can synthesize phosphatides independently or whether it acquires phosphatides from the plasma only after their formation by a more active tissue, was answered by Chaikoff et al. (62) in the following manner. A sciatic nerve of a dog stripped free of all adipose and connective tissue and weighing 300 mg. was placed in 5 ml. of Ringer solution containing radioactive phosphate. For control purposes the adipose-connective tissue surrounding the nerve was treated in a similar way. Conversion of radiophosphate from the Binger solution into radiophosphatide by the nerve was found to be considerable, as is seen in Table XVIII. These experiments show that the nerve process, separated from the nerve cell body, can form phosphatides from inorganic phosphate. 

Glycerophosphate and phosphoryl ethanolamine containing were prepared by Chaikoff et al, (32), and their incorporation into phosphatides of liver and kidney was demonstrated by surviving slices and also in the intact animal. These experiments gave no proof that breakdown of these labeled compounds to inorganic phosphate did not occur before conversion of the radioactive phosphorus to phosphatide. 

Within the yolk, no new formation of phosphatides (no formation of labeled phosphatides) takes place. This is shown by the fact that, if the labeled phosphate is administered after the egg has left the ovary, no active phosphatides are found in the yolk, as distinct from active inorganic phosphate which penetrates from the circulation into the egg during every phase of its formation. 

The effect of Roentgen rays on turnover rate of phosphatides present both in tissue and in nuclei was investigated as well. Two groups of twelve rats, after irradiation with 1000 r, are given labeled phosphate, while nonirradiated, control groups are treated in a similar way. After the lapse of two hours the animals are sacrificed and the sarcoma and livers are pooled separately. An aliquot is used in the determination of specific activities of inoi anic and phosphatide phosphorus of the tissue, while from the bulk of the material cell nuclei are isolated by the method of Bounce (42). The specific activities of the corresponding phosphorus fractions of the nuclei are also determined, and furthermore the activity of the inorganic phosphorus of the pooled blood plasma is measured. As seen in Tables XXXI and XXXII, the rate of turnover of phosphatides in liver... 

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