Enzymes Of Phospholipid Synthesis Research Articles

Synthesis of phospholipids in human placenta

IntroductionPlacental phospholipid synthesis is critical for the expansion of the placental exchange surface area and for production of signaling molecules. Despite their importance, it is not yet established which enzymes involved in the de novo synthesis and remodeling of placental phospholipids are expressed and active in the human placenta. MethodsWe identified phospholipid synthesis enzymes by immunoblotting in placental homogenates and immunofluorescence in placenta tissue sections. Primary human trophoblast (PHT) cells from term healthy placentas (n = 10) were cultured and exposed to 13C labeled fatty acids (16:0, 18:1 and 18:2 n-6, 22:6 n-3) for 2 and 24 h. Three phospholipid classes; phosphatidic acid, phosphatidylcholine, and lysophosphatidylcholine containing 13C fatty acids were quantified by Liquid Chromatography with tandem mass spectrometry (LC/MS–MS). ResultsAcyl transferase and phospholipase enzymes were detected in human placenta homogenate and primarily expressed in the syncytiotrophoblast. Three representative 13C fatty acids (16:0, 18:1 and 18:2 n-6) were incorporated rapidly into phosphatidic acid in trophoblasts, but 13C labeled docosahexaenoic acid (DHA; 22:6 n-3) incorporation was not detected. 13C DHA was incorporated into phosphatidylcholine. Lysophosphatidylcholine containing all four 13C labeled fatty acids were found in high abundance. ConclusionsPhospholipid synthesis and remodeling enzymes are present in the syncytiotrophoblast. 13C labeled fatty acids were rapidly incorporated into cellular phospholipids. 13C DHA was incorporated into phospholipids through the remodeling pathway rather than by de novo synthesis. These understudied pathways are highly active and critical for structure and function of the placenta.

Pseudomonas aeruginosa responds to altered membrane phospholipid composition by adjusting the production of two-component systems, proteases and iron uptake proteins

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. To achieve these, bacteria use adaptation mechanisms involving covalent modification and remodelling of the acyl chain length of PLs. However, little is known about bacterial pathways regulated by PLs. Here, we investigated proteomic changes in the biofilm of P. aeruginosa phospholipase mutant (∆plaF) with altered membrane PL composition. The results revealed profound alterations in the abundance of many biofilm-related two-component systems (TCSs), including accumulation of PprAB, a key regulator of the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of several proteases, in ∆plaF, indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional response. Moreover, proteomics and biochemical assays revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in ∆plaF, while proteins from alternative iron-uptake systems were accumulated. These suggest that PlaF may function as a switch between different iron-acquisition pathways. The observation that PL-acyl chain modifying and PL synthesis enzymes were overproduced in ∆plaF reveals the interconnection of degradation, synthesis and modification of PLs for proper membrane homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that alteration of PL composition in ∆plaF plays a role for the global adaptive response in P. aeruginosa mediated by TCSs and proteases. Our study revealed the global regulation of virulence and biofilm by PlaF and suggests that targeting this enzyme may have therapeutic potential.

Dysregulation of Lipid Metabolism in Macrophages Is Responsible for Severe Endotoxin Tolerance in FcgRIIB-Deficient Lupus Mice.

FcgRIIB dysfunction is commonly found in patients with lupus, especially in Asia. LPS-tolerance is prominent in FcgRIIB–/– lupus mice. LPS-tolerant macrophages demonstrate cell energy depletion, which might affect lipid metabolism. Therefore, to explore lipid metabolism, LPS-tolerance was induced twice by LPS administration in macrophages and in mice. LPS-tolerant FcgRIIB–/– macrophages demonstrated lesser mitochondrial DNA (mtDNA), more severe ATP depletion, lower cytokine production, and higher lipid accumulation (oil red O staining) compared to LPS-tolerant WT cells. Mass-spectrometry-based lipidomic analysis demonstrated a higher abundance of phosphatidylethanolamine (PE) phospholipid in LPS-tolerant FcgRIIB–/– macrophages than WT cells. This was at least in part due to the lower expression of phosphatidylethanolamine N-methyltransferase (pemt), an enzyme that converts PE to phosphatidylcholine (PC). Aminoimidazole-4-carboxamide ribonucleotide (AICAR), a pemt inhibitor, worsens LPS-tolerance in WT macrophages and supports the impact of pemt upon LPS-tolerant FcgRIIB–/– macrophages. Additionally, phosphorylated AMP-activated protein kinase (AMPK-p), a molecule for ATP-restoration associated with pemt, and phosphorylated acetyl CoA carboxylase, a downstream signaling of AMPK-p, were higher in LPS-tolerant FcgRIIB–/– macrophages than WT. Furthermore, Compound C, an AMPK inhibitor, attenuated LPS-tolerance in both FcgRIIB–/– macrophages and mice. Taken together, the intense decrease in cytokine production after the second LPS stimulation (LPS-tolerance) in FcgRIIB–/– macrophages was possibly due to the impact of an immense cytokine synthesis after the first dose of LPS. This includes using up PEMT, an enzyme of phospholipid synthesis during cytokine production, and AMPK-p induction in response to profound ATP-depletion. Therefore, the manipulation of the AMPK/PEMT axis provides a novel therapeutic candidate for the treatment of severe LPS-tolerance in lupus.

The Long Hunt for pssR-Looking for a Phospholipid Synthesis Transcriptional Regulator, Finding the Ribosome.

The phospholipid (PL) composition of bacterial membranes varies as a function of growth rate and in response to changes in the environment. While growth adaptation can be explained by biochemical feedback in the PL synthesis pathway, recent transcriptome studies have revealed that the expression of PL synthesis genes can also be tuned in response to various stresses. We previously showed that the BasRS two-component pathway controls the expression of the diacylglycerol kinase gene, dgkA, in Escherichia coli (A. Wahl, L. My, R. Dumoulin, J. N. Sturgis, and E. Bouveret, Mol Microbiol, 80:1260-1275, 2011, https://doi.org/10.1111/j.1365-2958.2011.07641.x). In this study, we set up a strategy to identify the mutation responsible for the upregulation of pssA observed in the historical pssR1 mutant and supposedly corresponding to a transcriptional repressor (C. P. Sparrow and J. Raetz, J Biol Chem, 258:9963-9967, 1983). pssA encodes phosphatidylserine synthase, the first step of phosphatidylethanolamine synthesis. We showed that this mutation corresponded to a single nucleotide change in the anti-Shine-Dalgarno sequence of the 16S rRNA encoded by the rrnC operon. We further demonstrated that this mutation enhanced the translation of pssA Though this effect appeared to be restricted to PssA among phospholipid synthesis enzymes, it was not specific, as evidenced by a global effect on the production of unrelated proteins.IMPORTANCE Bacteria adjust the phospholipid composition of their membranes to the changing environment. In addition to enzymatic regulation, stress response regulators control specific steps of the phospholipid synthesis pathway. We wanted to identify a potential regulator controlling the expression of the phosphatidylserine synthase gene. We showed that it was not the previously suggested hdfR gene and instead that a mutation in the anti-Shine-Dalgarno sequence of 16S RNA was responsible for an increase in pssA translation. This example underlines the fact that gene expression can be modulated by means other than specific regulatory processes.

Effect of Fabp1/Scp-2/Scp-x Ablation on Whole Body and Hepatic Phenotype of Phytol-Fed Male Mice.

Liver fatty acid binding protein (Fabp1) and sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) genes encode proteins that enhance hepatic uptake, cytosolic transport, and peroxisomal oxidation of toxic branched-chain fatty acids derived from dietary phytol. Since male wild-type (WT) mice express markedly higher levels of these proteins than females, the impact of ablating both genes (TKO) was examined in phytol-fed males. In WT males, high phytol diet alone had little impact on whole body weight and did not alter the proportion of lean tissue mass (LTM) versus fat tissue mass (FTM). TKO conferred on dietary phytol the ability to induce weight loss as well as reduce liver weight, FTM, and even more so LTM. Concomitantly TKO induced hepatic lipid accumulation, preferentially threefold increased phospholipid (PL) at the expense of decreased triacylglycerol (TG) and total cholesterol. Increased PL was associated with upregulation of membrane fatty acid transport/translocase proteins (FATP 2,4), cytosolic fatty acid/fatty acyl-CoA binding proteins (FABP2, ACBP), and the rate limiting enzyme in PL synthesis (Gpam). Decreased TG and cholesterol levels were not attributable to altered levels in respective synthetic enzymes or nuclear receptors. These data suggest that the higher level of Fabp1 and Scp2/Scpx gene products in WT males was protective against deleterious effects of dietary phytol, but TKO significantly exacerbated phytol effects in males.

Revisiting the cell biology of the acyl-ACP:phosphate transacylase PlsX suggests that the phospholipid synthesis and cell division machineries are not coupled in Bacillus subtilis.

PlsX is a central enzyme of phospholipid synthesis in bacteria, converting acyl-ACP to acyl-phosphate on the pathway to phosphatidic acid formation. PlsX has received attention because it plays a key role in the coordination of fatty acid and phospholipid synthesis. Recently, PlsX was also suggested to coordinate membrane synthesis with cell division in Bacillus subtilis. Here, we have re-investigated the cell biology of PlsX and determined that the enzyme is uniformly distributed on the membrane of most cells, but occasionally appears as membrane foci as well. Foci and homogenous patterns seem freely interconvertible but the prevalence of the uniform staining suggests that PlsX does not need to localize to specific sites to function correctly. We also investigated the relationship between PlsX and the divisome. In contrast to previous observations, PlsX's foci showed no obvious periodicity of localization and did not colocalize with the divisome. Furthermore, depletion of PlsX did not affect cell division if phospholipid synthesis is maintained by an alternative enzyme. These results suggest that coordination between division and membrane synthesis may not require physical or functional interactions between the divisome and phospholipid synthesis enzymes.

Mechanistic insights into the nongenomic regulation of phospholipid synthesizing enzymes

Lipid synthesis is a complex process regulated at multiple levels. Here, we will discuss nongenomic regulatory mechanisms, particularly the activation and/or recruitment of key enzymes to membranes. The phospholipid synthesis enzymes Lipin and CTP:phosphocholine cytidylyltransferase are taken as examples of these mechanisms that are mediated by posttranslational modifications or by an intrinsic property of the enzyme that senses lipid composition. In addition, special emphasis will be put on another relevant non genomic lipid synthesis regulation mechanism that is dependent on c-Fos, a protein that has deserved less attention so far. This latter regulatory mechanism is emerging as an important determinant for processes that require high rates of lipid synthesis such as those of growth and proliferation.

Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Regulation of Phospholipid Synthesis in the Yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae, with its full complement of organelles, synthesizes membrane phospholipids by pathways that are generally common to those found in higher eukaryotes. Phospholipid synthesis in yeast is regulated in response to a variety of growth conditions (e.g., inositol supplementation, zinc depletion, and growth stage) by a coordination of genetic (e.g., transcriptional activation and repression) and biochemical (e.g., activity modulation and localization) mechanisms. Phosphatidate (PA), whose cellular levels are controlled by the activities of key phospholipid synthesis enzymes, plays a central role in the transcriptional regulation of phospholipid synthesis genes. In addition to the regulation of gene expression, phosphorylation of key phospholipid synthesis catalytic and regulatory proteins controls the metabolism of phospholipid precursors and products.

Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli

Phospholipid homeostasis of the bacterial membrane is maintained by biochemical regulation of the synthesis enzymes depending on the environment. However, genes encoding phospholipid synthesis enzymes might also be regulated during stress responses, in order for the bacteria to adapt their growth to changing environments. While few studies have addressed this question, global analyses show that specific genes are activated by alternative Sigma factors, and that phospholipid synthesis genes are co-ordinately regulated during stringent response. In Escherichia coli, the genes coding for glycerol-3-phosphate acyltransferase and diacylglycerol kinase (plsB and dgkA) are found next to each other in divergent orientations, suggesting a co-ordinated regulation. We investigated their regulation and found that these two genes are inversely regulated by a diversity of stress responses. plsB activation by σE is concomitant with a reduced DgkA amount. A second proximal promoter for plsB expression is responsible for basal plsB expression and is inhibited during stringent response. Finally, dgkA is activated by the two-component regulator BasR, linking dgkA function of phospholipid recycling to LPS modifications. In E. coli, PlsB and DgkA are key enzymes in the phospholipid synthesis pathway. Our results show that their expression is a crucial point of integration for different stress signals.

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A

Phosphatidylserine (PS) synthase catalyzes the committed step of phosphatidylcholine synthesis via the CDP‐diacylglycerol pathway in the yeast Saccharomyces cerevisiae. It is one of the most highly regulated phospholipid synthesis enzymes in yeast. Previous work showed that PS synthase is phosphorylated by protein kinase A, and that phosphorylation inhibited enzyme activity. In this work, the sites of protein kinase A phosphorylation were examined. Upon SDS‐PAGE, the PS synthase migrates as a doublet of two protein bands. Protein kinase A phosphorylation of the native enzyme resulted in the disappearance of the lower protein band, whereas treatment with alkaline phosphatase or protein phosphatase resulted in the disappearance of the upper protein band. A site‐directed mutagenesis analysis of putative protein kinase A sites (Ser46, Ser47, Ser140, Ser141) revealed that Ser46 and Ser47 were the major phosphorylation sites in the enzyme. A double S46A S47A mutation resulted in an enzyme that migrated on SDS‐gels as a single protein band, whereas single S46A and S47A mutant proteins migrated as doublets with a decrease in the abundance of the upper band. PS synthase was also subject to proteolytic degradation, and the phosphorylation of the enzyme at Ser46 and Ser47 protected the enzyme from proteolysis. Supported by NIH grant GM 50679.

Biosynthesis of Membrane Lipids.

The pathways in Escherichia coli and (largely by analogy) S. enterica remain the paradigm of bacterial lipid synthetic pathways, although recently considerable diversity among bacteria in the specific areas of lipid synthesis has been demonstrated. The structural biology of the fatty acid synthetic proteins is essentially complete. However, the membrane-bound enzymes of phospholipid synthesis remain recalcitrant to structural analyses. Recent advances in genetic technology have allowed the essentialgenes of lipid synthesis to be tested with rigor, and as expected most genes are essential under standard growth conditions. Conditionally lethal mutants are available in numerous genes, which facilitates physiological analyses. The array of genetic constructs facilitates analysis of the functions of genes from other organisms. Advances in mass spectroscopy have allowed very accurate and detailed analyses of lipid compositions as well as detection of the interactions of lipid biosynthetic proteins with one another and with proteins outside the lipid pathway. The combination of these advances has resulted in use of E. coli and S. enterica for discovery of new antimicrobials targeted to lipid synthesis and in deciphering the molecular actions of known antimicrobials. Finally,roles for bacterial fatty acids other than as membrane lipid structural components have been uncovered. For example, fatty acid synthesis plays major roles in the synthesis of the essential enzyme cofactors, biotin and lipoic acid. Although other roles for bacterial fatty acids, such as synthesis of acyl-homoserine quorum-sensing molecules, are not native to E. coli introduction of the relevant gene(s) synthesis of these foreign molecules readily proceeds and the sophisticated tools available can used to decipher the mechanisms of synthesis of these molecules.

Phosphorylation of the Yeast Phospholipid Synthesis Regulatory Protein Opi1p by Protein Kinase A

The Opi1p transcription factor plays a negative regulatory role in the expression of UASINO-containing genes involved in phospholipid synthesis in the yeast Saccharomyces cerevisiae. The phosphorylation of Opi1p by protein kinase A (cAMP-dependent protein kinase) was examined in this work. Using a maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase A activity was time- and dose-dependent and dependent on the concentrations of Opi1p and ATP. Protein kinase A phosphorylated Opi1p on multiple serine residues. The synthetic peptides SCRQKSQPSE and SQVRESLLNL containing the protein kinase A motif for Ser31 and Ser251, respectively, within Opi1p were substrates for protein kinase A. Phosphorylation of S31A and S251A mutant maltose-binding protein-Opi1p fusion proteins by protein kinase A was reduced when compared with the wild type protein, and phosphopeptides present in wild type Opi1p were absent from the S31A and S251A mutant proteins. In vivo labeling experiments showed that the extent of phosphorylation of the S31A and S251A mutant proteins was reduced when compared with the wild type protein. The physiological consequence of the phosphorylation of Opi1p at Ser31 and Ser251 was examined by measuring the effects of the S31A and S251A mutations on the expression of the UASINO-containing gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacZ reporter gene in opi1Delta mutant cells expressing the S31A and S251A mutant Opi1p proteins was elevated 42 and 35%, respectively, in the absence of inositol and 55 and 52%, respectively, in the presence of inositol when compared with cells expressing wild type Opi1p. These data supported the conclusion that phosphorylation of Opi1p at Ser31 and Ser251 mediated the stimulation of the negative regulatory function of Opi1p on the expression of the INO1 gene.

Effect of epidermal growth factor on enzymes of phospholipid biosynthesis in lung and liver of fetal rat in vivo and in vitro.

Epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, is an important regulator of cell differentiation and fetal lung surfactant synthesis, and may be used as a potential novel therapeutic agent in prematurity. Nevertheless, the distinct role in lung development and its mechanisms of action are not well understood. We investigated in vivo the systemic effect of intrafetally administered EGF (200 ng/g fetal body weight) and maternally administered dexamethasone (DEXA; 0.2 and 2.0mg/kg maternal body weight) on the activity of important enzymes of the phospholipid synthesis in the fetal rat lung and liver: choline kinase (EC 2.7.1.32), cholinephosphate cytidyltransferase (EC 2.7.7.15), choline phosphotransferase (EC 2.7.8.2), lysolecithin acyltransferase (EC 2.3.1.23) and glycerolphosphate phosphatidyltransferase (EC 2.7.8.5). Additionally, in vivo and in vitro effects of DEXA on EGF receptor synthesis, and the effects of EGF on protein content and morphogenesis of the fetal rat lung organoid culture, were evaluated. Whereas DEXA induced the activity of all investigated enzymes of phospholipid synthesis and increased EGF receptor synthesis, EGF has no effects on the enzymes, either in vivo or in vitro. EGF enhanced protein synthesis and morphogenesis in vitro. With respect to our data and the literature, we hypothesize that DEXA and EGF may act on different cellular sides. Whereas glucocorticoids induce surfactant phospholipid synthesis, EGF should be more involved in cell proliferation and morphogenesis.

Low activity of key phospholipid catabolic and anabolic enzymes in human substantia nigra: possible implications for Parkinson's disease

To determine whether increased oxidative stress in substantia nigra of patients with idiopathic Parkinson's disease might be related to decreased ability of nigral cells to detoxify oxidized membrane phospholipids, we compared levels of the major phospholipid metabolizing enzymes in autopsied substantia nigra with those in non-nigral ( n=11) brain areas of the normal human brain. Whereas most enzymes possessed a relatively homogeneous distribution, the activity of the major phospholipid catabolizing enzyme phospholipase A 2, assayed in the presence of calcium ions, varied amongst different regions, with substantia nigra possessing the lowest activity. Similarly, calcium-independent phospholipase A 2 activity, although possessing a relatively homogeneous regional distribution, was also low in the substantia nigra. This, coupled with low activity of phosphoethanolamine- and phosphocholine-cytidylyltransferases, major regulatory enzymes of phospholipid synthesis, in this brain region, suggest that the rate of phospholipid turnover is low in the substantia nigra. Low activity of key phospholipid catabolic and anabolic enzymes in human substantia nigra might result in reduced ability to repair oxidative membrane damage, as may occur in Parkinson's disease.

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.

Role of cyclic adenosine monophosphate in phospholipid synthesis in Mycobacterium smegmatis ATCC 607.

The present study was undertaken to examine the influence of intracellular levels of cyclic AMP on phospholipid synthesis in Mycobacterium smegmatis ATCC 607. The cyclic AMP levels were modulated by growing cells in the presence of activator (forskolin) and inhibitor (atropine) of adenylate cyclase, the synthesizing enzyme of cyclic AMP. Forskolin-grown cells exhibited a 1.4-fold increase in the level of cAMP while a similar decrease (1.8-fold) was seen with atropine-grown cells. These altered levels of cAMP in turn affected the total content, composition and synthesis of phospholipids. Total phospholipid content increased and decreased in cells grown in the presence of forskolin and atropine, respectively. These observations were further supported by alterations in [14C]acetate incorporation as well as in activities of glycerol kinase and glycerol-3-phosphate acyltransferase, the key enzymes of phospholipid synthesis. Protein phosphorylation mechanism seems to be involved in phospholipid metabolism as the activities of protein kinase increased and decreased in cells grown in forskolin or atropine cells. Our results demonstrate a correlation between phospholipid synthesis and intracellular levels of cAMP in M. smegmatis.

Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria

In the yeast, Saccharomyces cerevisiae, similar to higher eukaryotes most phospholipids are synthesized in microsomes. Mitochondria contribute to the cellular biosynthesis of phospholipids insofar as they harbor phosphatidylethanolamine decarboxylase, and enzymes of phosphatidylglycerol and cardiolipin synthesis. In this paper we present evidence that certain enzymes of phospholipid biosynthesis, namely phosphatidylserine and phosphatidylinositol synthase, are enriched in a special microsomal fraction associated with mitochondria, which we named MAM. This fraction was isolated and characterized with respect to marker enzymes, protein and phospholipid composition, and enzymes of phospholipid synthesis. According to these analyses MAMs are a specialized subfraction of the endoplasmic reticulum, which is distinct from other microsomal subfractions. Phosphatidylserine synthesized in MAMs can be readily imported into mitochondria and converted to phosphatidylethanolamine. Reassociation of MAMs with purified mitochondria led to reconstitution of the import of phosphatidylserine into mitochondria. Organelle contact is suggested as a possible mechanism of this process.