Newly imported ethanolamine is preferentially utilized for phosphatidylethanolamine biosynthesis in the hamster heart.

The effects of exogenous ethanolamine concentrations on ethanolamine uptake and its subsequent incorporation into phosphatidylethanolamine were examined. Hamster hearts were perfused with 0.04-1000 microM labelled ethanolamine. Analysis of radioactivity distribution in ethanolamine-containing metabolites revealed an accumulation of labelled ethanolamine when the heart was perfused with greater than or equal to 0.4 microM labelled ethanolamine. The changes in radioactivity distribution indicated that the phosphorylation of ethanolamine had become rate-limiting in the CDP-ethanolamine pathway when the heart was perfused with greater than or equal to 0.4 microM ethanolamine. Perfusion with different concentrations of ethanolamine did not significantly change the intracellular ethanolamine pool. The accumulation of labelled ethanolamine without a corresponding change in the ethanolamine pool suggests that the newly imported ethanolamine did not equilibrate with the endogenous ethanolamine pool. We postulate that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis.     

Modulation of phosphatidylethanolamine biosynthesis by exogenous ethanolamine and analogues in the hamster heart

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations ( 0.1 M), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine ( 0.4 M) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine.The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine. (Mol Cell Biochem116: 69–73, 1992)     

Inhibition of phosphatidylethanolamine biosynthesis in baby hamster kidney-21 cells infected with Semliki Forest virus.

Semliki Forest virus inhibits phosphatidylethanolamine biosynthesis in baby hamster kidney-21 cells 6 h after infection. Viral infection reduced the incorporation of [1,2-14C]-ethanolamine into intact cells by approximately 50%. A similar reduction in the activity of the ethanolaminephosphotransferase (EC 2.7.8.1) was also observed. The apparent Km for CDPethanolamine was 60 muM for the microsomal enzymes from infected or mock-infected cells. In addition, exogenous diglyceride only stimulated by 1.5-fold the ethanolaminephosphotransferase from virus- or mock-infected cells, whereas the same diglyceride preparations stimulated the cholinephosphotransferase (EC 2.7.8.2) from baby hamster kidney cells by sixfold. Generation of endogenous diglyceride by pretreatment of the microsomes with phospholipase C (EC 3.1.4.3) stimulated the activity of the cholinephosphotransferase but not the ethanolaminephosphotranferase. Semliki Forest virus does not inhibit all microsomal enzymes, since the activities of NADH- K3Fe(CN)6 reductase and NADH dehydrogenase (EC 1.6.99.3) were not affected. The ethanolaminephosphotransferase from virus- and mock-infected cells showed similar profiles of activity as a function of temperature; this result and other studies suggest that that membranous environment of the ethanolaminephosphotransferase was not significantly modified by the virus.     

Characterization of the pathways for phosphatidylethanolamine biosynthesis in Chinese hamster ovary mutant and parental cell lines

A tritium suicide procedure was devised to facilitate the isolation of Chinese hamster ovary cell mutants defective in phosphatidylethanolamine biosynthesis. One mutant with a 20-50% reduction in [3H]ethanolamine incorporation was chosen for further analysis and was shown to have reduced activity of CTP: phosphoethanolamine cytidylyltransferase. Levels of phosphatidylethanolamine and rates of its biosynthesis were compared in the mutant and parent cell lines. Despite the reduced activity of the CDP-ethanolamine pathway in the mutant, levels of phosphatidylethanolamine were the same in mutant and parent cells. Rates of phosphatidylethanolamine synthesis de novo, as measured by incorporation of 32PO4 into phosphatidylethanolamine, were also the same in mutant and parent cells, as was the rate of incorporation of [3H]serine into both phosphatidylserine and phosphatidylethanolamine. After a long term labeling with [3H]serine, the specific radioactivity of phosphatidylserine was the same as that of phosphatidylethanolamine, and there was no difference in the specific radioactivities of the two lipids between mutant and parent cells. These results implicate decarboxylation of phosphatidylserine as the sole route for synthesis of phosphatidylethanolamine under normal culture conditions.     

Okadaic acid inhibits phosphatidylethanolamine biosynthesis in rat hepatocytes.

Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A, inhibited the synthesis of phosphatidylethanolamine via the CDPethanolamine pathway in isolated hepatocytes. Pulse-chase experiments and measurement of the enzyme activity demonstrated that the inhibition of phosphatidylethanolamine synthesis was not caused by an inhibition of CTP:phosphoethanolamine cytidylyltransferase, the putative regulatory enzyme. However, okadaic acid decreased the cellular diacylglycerol level to 30% of that in control cells. The data suggest that the availability of diacylglycerol limits phosphatidylethanolamine synthesis in okadaic acid-treated hepatocytes.     

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors

In the preceding paper, we reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [32P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [32P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.     

Effects of fatty acids on phosphatidylcholine biosynthesis in isolated hamster heart

The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid.     

Abortive infection with Sindbis virus of a Chinese hamster ovary cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis

The effects of phosphatidylserine starvation on the infection with Sindbis virus (an enveloped RNA virus) have been investigated in a Chinese hamster ovary (CHO) cell mutant (strain PSA-3) which requires exogenously added phosphatidylserine for cell growth because it lacks the ability to synthesize this phospholipid. When PSA-3 cells were grown in the absence of phosphatidylserine, the cellular contents of phosphatidylserine and also phosphatidylethanolamine produced through decarboxylation of phosphatidylserine decreased. Sindbis virus production in the mutant cells decreased immediately upon phosphatidylserine deprivation as did the contents of phosphatidylserine and phosphatidylethanolamine, whereas the cell growth, viability, and syntheses of protein, DNA and RNA remained normal for approx. 40 h phosphatidylserine starvation. Although PSA-3 cells grown without phosphatidylserine for 24 h were able to bind and internalize Sindbis virus almost normally, viral RNA synthesis was greatly reduced in the cells, suggesting that nucleocapsids of internalized Sindbis virus are not normally released into the cytoplasm. Unlike mammalian cell mutants defective in endosomal acidification, PSA-3 cells grown without phosphatidylserine were not resistant to diphtheria toxin. Furthermore, the yield of virions and viral RNA synthesis in PSA-3 cells were not completely restored on brief exposure of the cells to low pH medium following virus adsorption, which is known to induce artificial fusion of the viral envelope with the plasma membrane of normal host cells and then injection of viral nucleocapsids into the cytoplasm. Our data demonstrate the requirement of membrane phospholipids, such as phosphatidylserine and/or phosphatidylethanolamine, in CHO cells for Sindbis virus infection, and we discuss their possible roles.     

Serine phosphorylation negatively regulates RhoA in vivo

Previous work indicates that RhoA phosphorylation on Ser188 by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser188 upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.     

Serine transhydroxymethylase in methionine biosynthesis in Saccharomyces cerevisiae

Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.     

Headgroup biosynthesis of phosphatidylcholine and phosphatidylethanolamine in seed plants

Phospholipid biosynthesis is crucial for plant growth and development. It involves attachment of fatty acids to a phospho-diacylglycerol backbone and modification of the phospho-group into an amino alcohol. The biochemistry and molecular biology of the former has been well established, but a number of enzymes responsible for the latter have only recently been cloned and functionally characterized in Arabidopsis and some other model plant species. The metabolism involving the polar head groups of phospholipids established by past biochemical studies can now be validated by available gene knockout models. Moreover, gene knockout studies have revealed emerging functions of phospholipids in regulating plant growth and development. This review aims to revisit the old questions of polar headgroup biosynthesis of plant phosphatidylcholine and phosphatidylethanolamine by giving an overview of recent advances in the field and beyond.     

Isolation of somatic cell mutants defective in the biosynthesis of phosphatidylethanolamine

An in situ autoradiographic assay for CDP-ethanolamine:1,2-sn-diacylglycerol ethanolamine phosphotransferase (EC 2.7.8.1) activity in Chinese hamster ovary cells was developed and used to screen approximately 10,000 individual mutagen-treated colonies attached to filter paper (Esko, J. D., and Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1190-1193). A variant (strain 40.11) was isolated in which the ethanolamine phosphotransferase specific activity in vitro was 6-10-fold less than in the parent, but the level of CDP-choline:1,2-sn-diacylglycerol choline phosphotransferase (EC 2.7.8.2) activity was normal. In extracts, the mutant was also defective in the synthesis of ethanolamine plasmalogen. In vivo, the short term kinetics of labeling with [32P]phosphate or [14C]ethanolamine was correspondingly altered. However, the long tem growth rate and steady state phospholipid compositions of the mutant and parent were quite similar. These results show that the ethanolamine and choline phosphotransferases of Chinese hamster ovary cells are distinct as judged by genetic criteria, while the biosynthesis of phosphatidylethanolamine and its plasmalogen share common enzymatic component(s).     

Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (greater than or equal to 0.1 mM), choline uptake was enhanced 20-38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.     

Acyl specificity of hamster heart CDP-choline 1,2-diacylglycerol phosphocholine transferase in phosphatidylcholine biosynthesis

The acyl specificity of 1,2-diacylglycerol: CDP-choline phosphocholine transferase (EC 2.7.8.2) for the formation of phosphatidylcholine with the appropriate acyl groups in hamster heart was investigated. Enzyme activity was determined in the microsomal fraction with 1,2-diacylglycerols of known acyl content. Maximum enzyme activity was obtained with diacylglycerol containing a monoenoic acyl group at the C-2 position of the glycerol moiety, regardless of the acyl group at the C-1 position. The specificity of the enzymes was also investigated by perfusing the isolated hamster heart with labelled glycerol. Comparison of the molecular species of the labelled diacylglycerols and phosphatidylcholine subsequent to perfusion revealed that the specificity of phosphocholine transferase was not limited to the monoenoic species of diacylglycerol. The difference in specificity observed between the in vitro assay and the perfusion study may partly be attributed to the presence of detergent in the enzyme assay mixture (to facilitate solubility of diacylglycerol). It is concluded that in the hamster heart, phosphocholine transferase has only limited ability to select the appropriate acyl groups for phosphatidylcholine biosynthesis. It appears that the majority of the newly formed phosphatidylcholine in the heart via the CDP-choline pathway is subsequently resynthesized by deacylation-reacylation process.     

Serine biosynthesis and its regulation in Bacillus subtilis

Cell-free extracts of Bacillus subtilis strains GSY and 168 convert (14)C-phosphoglycerate to (14)C-serine phosphate and (14)C-serine. These reactions indicate a functional phosphorylated pathway for serine biosynthesis in these cells. The addition of serine to the incubation mixture inhibited the formation of both radioactive products. Extracts of mutant strains that require serine for growth lacked the capacity to synthesize serine phosphate, confirming that the phosphorylated pathway was the only functional pathway available for serine synthesis. Serine phosphate phosphatase and phosphoglycerate dehydrogenase activity were demonstrated in cell extracts, and the phosphoglycerate dehydrogenase was shown to be inhibited specifically by l-serine. The extent of serine inhibition increased when the temperature was raised from 25 to 37 C, and the thermal stability of the enzyme was enhanced by the presence of the inhibitor serine or the coenzyme reduced nicotinamide adenine dinucleotide. At 37 C the curve representing the relationship between phosphoglycerate concentration and enzyme velocity was biphasic, and the serine inhibition which was competitive at low substrate concentrations became noncompetitive at higher concentrations.     

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat-2 fibroblasts by cell-permeable ceramides.

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.