Rate Of Phosphatidylethanolamine Synthesis Research Articles

The phosphatidylethanolamine level of yeast mitochondria is affected by the mitochondrial components Oxa1p and Yme1p

The majority of phosphatidylethanolamine, an essential component of yeast mitochondria, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p), a component of the inner mitochondrial membrane. Here, we report that deletion of OXA1 encoding an inner mitochondrial membrane protein translocase markedly affects the mitochondrial phosphatidylethanolamine level. In an oxa1Delta mutant, cellular and mitochondrial levels of phosphatidylethanolamine were lowered similar to a mutant with PSD1 deleted, and the rate of phosphatidylethanolamine synthesis by decarboxylation of phosphatidylserine in vivo and in vitro was decreased. This was due to a lower PSD1 transcription rate in the oxa1Delta mutant compared with wild-type and compromised assembly of Psd1p into the inner mitochondrial membrane. Lack of Mba1p, another component involved in the assembly of mitochondrial proteins into the inner mitochondrial membrane, did not affect the amount of phosphatidylethanolamine or the assembly of Psd1p. Deletion of the inner membrane protease Yme1p enhanced Psd1p stability suggesting that Yme1p contributed substantially to the proteolytic turnover of Psd1p in wild-type. In summary, our results demonstrate a link between the mitochondrial protein import machinery, assembly and stability of Psd1p, and phosphatidylethanolamine homeostasis in yeast mitochondria.

Phospholipid biosynthetic enzymes in human brain.

Growing evidence suggests an involvement of brain membrane phospholipid metabolism in a variety of neurodegenerative and psychiatric conditions. This has prompted the use of drugs (e.g., CDPcholine) aimed at elevating the rate of neural membrane synthesis. However, no information is available regarding the human brain enzymes of phospholipid synthesis which these drugs affect. Thus, the objective of our study was to characterize the enzymes involved, in particular, whether differences existed in the relative affinity of substrates for the enzymes of phosphatidylethanolamine (PE) compared to those of phosphatidylcholine (PC) synthesis. The concentration of choline in rapidly frozen human brain biopsies ranged from 32-186 nmol/g tissue, a concentration similar to that determined previously for ethanolamine. Since human brain ethanolamine kinase possessed a much lower affinity for ethanolamine (Km = 460 microM) than choline kinase did for choline (Km = 17 microM), the activity of ethanolamine kinase in vivo may be more dependent on substrate availability than that of choline kinase. In addition, whereas ethanolamine kinase was inhibited by choline, and to a lesser extent by phosphocholine, choline kinase activity was unaffected by the presence of ethanolamine, or phosphoethanolamine, and only weakly inhibited by phosphocholine. Phosphoethanolamine cytidylyltransferase (PECT) and phosphocholine cytidylyltransferase (PCCT) also displayed dissimilar characteristics, with PECT and PCCT being located predominantly in the cytosolic and particulate fractions, respectively. Both PECT and PCCT exhibited a low affinity for CTP (Km approximately 1.2 mM), suggesting that the activities of these enzymes, and by implication, the rate of phospholipid synthesis, are highly dependent upon the cellular concentration of CTP. In conclusion our data indicate different regulatory properties of PE and PC synthesis in human brain, and suggest that the rate of PE synthesis may be more dependent upon substrate (ethanolamine) availability than that of PC synthesis.

Phospholipid synthesis in the lymphomatous mouse liver studied by 31P nuclear magnetic resistance spectroscopy in vitro and by administration of 14C-radiolabelled compounds in vivo

High phosphomonoester to ATP ratios have been found in 31P magnetic resonance spectra from livers of patients with hepatic lymphoma (Dixon et al. (1990) Br. J. Cancer 63, 953–958). The present study of a murine lymphoma showed that the phosphomonoester in the lymphomatous liver was largely phosphoethanolamine, which is an intermediate of phospholipid metabolism. A significant positive correlation was found between the concentration of phosphoethanolamine, measured by high resolution 31P nuclear magnetic resonance spectroscopy of extracts, and the degree of infiltration, assessed by quantitative histology. The phosphoethanolamine concentration reached about 10 times its normal level, but the phosphocholine concentration remained the same as in the normal liver. Radiolabelling studies showed that while the rate of phosphoethanolamine synthesis from exogenous [ 14]ethanolamine was higher in the lymphomatous mouse liver than in control livers, the rate of phosphatidylethanolamine synthesis was lower in the lymphomatous liver. The rate of phosphatidylcholine synthesis in lymphoma-bearing livers was not significantly different from that in control mouse livers.

Phosphatidylethanolamine metabolism in rat liver after partial hepatectomy. Control of biosynthesis of phosphatidylethanolamine by the availability of ethanolamine.

The effect of partial (70%) hepatectomy on phosphatidylethanolamine (PE) synthesis was studied in rat liver during the first 4 post-operative days. Between 4 and 96 h after partial hepatectomy, the mass of PE increased from 30% to 80% of sham-operation values. In line with the increase in PE mass, the rate of PE synthesis in vivo from [14C]ethanolamine was stimulated 1.6- and 1.3-fold at 22 and 48 h after partial hepatectomy respectively. Surprisingly, the activity of CTP:phosphoethanolamine cytidylyltransferase (EC 2.7.7.14) was virtually unchanged after partial hepatectomy. In addition, neither ethanolamine kinase (EC 2.7.1.82) nor ethanolaminephosphotransferase (EC 2.7.8.1) showed any changes in activity over the time period studied. Hepatic levels of ethanolamine and phosphoethanolamine were drastically increased after partial hepatectomy, as compared with sham operation, whereas levels of CDP-ethanolamine and microsomal diacylglycerol were not affected. Interestingly, partial hepatectomy caused the concentration of free ethanolamine in serum to increase from 29 microM to approx. 50 microM during the first day after surgery. In hepatocytes isolated from non-operated animals, incorporation of [3H]ethanolamine into PE was stimulated by increasing the ethanolamine concentration from 10 up to 50 microM, whereas the radioactivity associated with phosphoethanolamine only increased at ethanolamine concentrations higher than 30 microM. Taken together, our results indicate that the observed increase in serum ethanolamine concentration after partial hepatectomy is probably responsible for both the increase in PE biosynthesis and the accumulation of ethanolamine and phosphoethanolamine in regenerating liver.

Biosynthesis of phosphatidylethanolamine via the CDP-ethanolamine route is an important pathway in isolated rat hepatocytes

In the present study pulse-label and pulse-chase experiments with isolated rat hepatocytes in suspension were designed to investigate the effects of the presence of either serine or ethanolamine in the medium on the rate of phosphatidylethanolamine synthesis via the CDPethanolamine pathway and by decarboxylation of phosphatidylserine. Addition of serine to the medium did not affect the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamine. Pulse-label experiments showed that the incorporation of [3H]serine into phosphatidylserine decreased in the presence of ethanolamine with a corresponding decrease of the incorporation of label into the ethanolamine base moiety of phosphatidylethanolamine. However, the radioactivity in the diacylglycerol part of phosphatidylethanolamine was considerably higher in the presence of ethanolamine than in its absence. Pulse-chase experiments with labelled serine demonstrated that the conversion of phosphatidylserine to phosphatidylethanolamine was not affected by varying concentrations of ethanolamine. Our observations indicate that in the presence of ethanolamine the biosynthesis of phosphatidylethanolamine via the CDPethanolamine pathway is enhanced relative to the synthesis by decarboxylation of phosphatidylserine.

Actions of dietary orotic acid on liver synthesis of phosphatidylcholine and phosphatidylethanolamine in rats.

The in vivo rates of the reactions of the cytidine pathways of liver phosphatidylcholine and phosphatidylethanolamine synthesis were measured in rats after 1 day of feeding on a semisynthetic diet containing 1% orotic acid. The calculations were made from the specific and total radioactivity versus time curves of the precursors and products following intraportal injection of [1,2-14C]choline, [2-14C]ethanolamine, and [2-3H]glycerol. The liver CTP level was increased twofold and the rates of CDP-choline and phosphatidylcholine synthesis were stimulated 4.5-fold in the rats fed orotic acid. The rate of CDP-ethanolamine synthesis was increased but could not be accurately quantified because of its extreme rapidity. No change occurred in the rate of the ethanolaminephosphotransferase reaction and the overall rate of phosphatidylethanolamine synthesis was unchanged by orotic acid feeding. The catalytic activities of the enzymes of the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis were not affected by feeding orotic acid for 1 day. Similar findings were obtained 3 h following intragastric administration of 100 mg of orotic acid. The results suggest the possibility that changes in the levels of liver CTP may play a role in regulation of the cytidine pathway of liver phosphatidylcholine synthesis but not of phosphatidylethanolamine synthesis, because the latter pathway appears to be tightly controlled at the ethanolaminephosphotransferase step.

Effect of 3,4-dihydroxybutyl-1-phosphonate on phosphoglyceride and lipoteichoic acid synthesis in Bacillus subtilis.

3,4-Dihydroxybutyl-1-phosphonate (CH2OHCHOHCH2CH2PO3HPO3H2), an analogue of glycerol 3-phosphate, preferentially inhibits the rate of synthesis and accumulation of phosphatidylglycerol in Bacillus subtilis W23 and 168. The rate of phosphatidylethanolamine synthesis is only slightly inhibited, whereas that of lysylphosphatidylglycerol is somewhat stimulated. As expected, decreased phosphatidylglycerol synthesis results in the inhibition of the formation of the putative lipoteichoic acid precursor, sn-glycero-1-phospho-beta-gentiobiosyldiacyglycerol and of lipoteichoic acid itself.

The effect of fasting and refeeding on the composition and synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in rat liver

Refeeding a high-sucrose, fat-free diet to fasted rats caused drastic alterations in the fatty acid composition of hepatic diacylglycerols, triacylglycerols, and phosphatidylcholines. However, the fatty acid profile of phosphatidylethanolamines did not change significantly. These results suggest that the fatty acid composition of diacylglycerols may influence the distribution of diacylglycerols among triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. Fasting and refeeding also affected the activities in vitro of a number of enzymes responsible for the formation of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. The activity of hepatic phosphatidate phosphatase increased fourfold upon refeeding. However, fasting the rats did not affect the activity of this enzyme despite the reduced triacylglycerol synthesis in the fasted liver in vivo. Fasting and refeeding induced alterations in the activities of diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase which correlated reasonably well with the changes observed in the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines in vivo, although the changes in diacylglycerol acyltransferase were too moderate. The changes in the activity of cholinephosphate cytidylyltransferase, which is suggested to catalyze the rate-limiting step in the formation of CDP-choline, ran parallel with the alterations in the synthesis of phosphatidylcholines in vivo. No such correlation was found between the activity of ethanolaminephosphate cytidylyltransferase and the rate of phosphatidylethanolamine synthesis. The present results indicate that the synthesis of triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines is controlled by the availability of the various substrates as well as by the activities of several enzymes involved in these processes.

Membrane phospholipid synthesis and phenotypic correlation of an Escherichia coli pss mutant

A pair of putatively isogenic pss(Ts) and pss+ (phosphatidylserine synthetase structural gene) strains was constructed and analyzed, together with the revertants, for the physiological consequences of cessation of the optimal synthesis of phosphatidylethanolamine (PE). Their in vivo and in vitro abilities to synthetize PE and the growth rates at different temperatures were determined. The rate of PE synthesis by OS2101 pss(Ts) was inversely related to the culture temperature. OS2101 in a low-salt broth medium stopped division and formed filamentous cells with declining viability upon the elevation of culture temperature from 27 to 42 or 44 degrees C, whereas the syntheses of deoxyribonucleic acid, ribonucleic acid, and protein were not affected. Proper concentrations of cations such as Na+, K+, NH4+, and Mg2+ or of sucrose could remedy the division and growth of OS2101 at the restrictive temperature without restoring normal PE synthesis. A remedial effect other than osmotic protection of these effectors and an adaptive regulatory mechanism for PE formation are suggested.

Regulation of phospholipid biosynthesis in isolated rat hepatocytes. Effect of different substrates.

The effects of choline, ethanolamine and its N-methyl analogs, different fatty acids, and L-methionine on phospholipid biosynthesis via the CDP-ester pathways and the methylation pathway were studied in rat hepatocytes. Phosphatidylethanolamine synthesis was stimulated severalfold by 0.02 to 0.1 mM ethanolamine, especially in the presence of long chain unsaturated fatty acids. At higher concentrations of ethanolamine, phosphorylethanolamine accumulated but the level of CDP-ethanolamine and the rate of phosphatidylethanolamine synthesis did not increase further. The rate of phosphatidylcholine synthesis via the CDP-ester pathway responded in a way analogous to that of phosphatidylethanolamine synthesis upon the addition of choline and fatty acid, except that a 10- to 20-fold higher concentration of choline was required for maximal stimulation, probably due to the rapid oxidation of choline to betaine. Phospholipids containing N-monomethyl- or N,N-dimethylethanolamine were efficiently formed from the corresponding free bases in the absence of ethanolamine and choline. Ethanolamine, but not other bases, inhibited completely phospholipid formation from N-monomethylethanolamine, probably as a result of competition at the level of CDP-ester formation. The data indicate that the cytidylytransferase reactions are rate-limiting steps in the synthesis of phosphatidylethanolamine and probably also phosphatidylcholine. In addition, the availability of diacylglycerol and its fatty acid composition may significantly affect the rate of phospholipid synthesis. The rate of phosphatidylcholine formation via phospholipid N-methylation approximately doubled when L-methionine was added at concentrations similar to that in rat plasma. Under these conditions the rate of phosphatidylcholine synthesis via this pathway was 20 to 40 percent of that via diacylglycerols and CDP-choline. The methylation of phosphatidylethanolamine to phosphatidylcholine remained essentially constant when the rate of phosphatidylethanolamine synthesis was varied 8-fold, but was significantly reduced when the formation of N-monomethyl- or N,N-dimethylphospholipid was stimulated by addition of the corresponding base. These phospholipids not only replaced phosphatidylethanolamine as the substrate for methylation but also increased the rate of phosphatidylcholine formation via this pathway. A method for the determination of nanomole amounts of different ethanolamine compounds is described.

The inhibition of phospholipid synthesis in Escherichia coli by phenethyl alcohol

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [ 32P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidylglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.