Phospholipid N-methyltransferase Research Articles

Global consequences of phosphatidylcholine reduction in Bradyrhizobium japonicum

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a limited number of bacteria including members of the Rhizobiales. Here, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum, respectively. Two different phospholipid N-methyltransferases, PmtA and PmtX1, convert phosphatidylethanolamine (PE) to PC by three consecutive methylation reactions in B. japonicum. PmtA mainly catalyzes the first methylation reaction converting PE to monomethyl PE, which then serves as substrate for PmtX1 performing the last two methylation reactions. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. A genome-wide survey for differentially expressed genes in the pmtA mutant with a custom-made Affymetrix gene chip revealed that PC reduction affects transcription of a strictly confined set of genes. Among the 11 up regulated genes were pmtX3 and pmtX4, which code for isoenzymes of PmtA. The expression of two typical two-component systems, a MarR-like regulator and two proteins of a RND-type (resistance nodulation cell division) efflux system were differentially expressed in the pmtA mutant. Our data suggests that a decrease in the PC content of B. japonicum membranes induces a rather specific transcriptional response involving three different transcriptional regulators all involved in the regulatory fine-tuning of a RND-type transport system.

Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants

The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.

Identification of novel suppressors for Mog1 implies its involvement in RNA metabolism, lipid metabolism and signal transduction

Mog1 is conserved from yeast to mammal, but its function is obscure. We isolated yeast genes that rescued a temperature-sensitive death of S. cerevisiae Scmog1Δ, and of S. pombe Spmog1 ts. Scmog1Δ was rescued by Opi3p, a phospholipid N-methyltransferase, in addition to S. cerevisiae Ran-homologue Gsp1p, and a RanGDP binding protein Ntf2p. On the other hand, Spmog1 ts was rescued by Cid13 that is a poly (A) polymerase specific for suc22 + mRNA encoding a subunit of ribonucleotide reductase, Ssp1 that is a protein kinase involved in stress response pathway, and Crp79 that is required for mRNA export, in addition to Spi1, S. pombe Ran-homologue, and Nxt2, S. pombe homologue of Ntf2p. Consistent with the identification of those suppressors, lack of ScMog1p dislocates Opi3p from the nuclear membrane and all of Spmog1 ts showed the nuclear accumulation of mRNA. Furthermore, SpMog1 was co-precipitated with Nxt2 and Cid13.

Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane

Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant-transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N-methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of host-microbe interactions.

The Yeast Phospholipid N-Methyltransferases Catalyzing the Synthesis of Phosphatidylcholine Preferentially Convert Di-C16:1 Substrates Both in Vivo and in Vitro

Phosphatidylcholine (PC) is an important and abundant structural component of the membranes of eukaryotic cells. In the yeast Saccharomyces cerevisiae, the primary route for the biosynthesis of PC consists of three consecutive methylation steps of phosphatidylethanolamine (PE) catalyzed by the phospholipid N-methyltransferases Cho2p and Opi3p. To investigate how these biosynthetic enzymes contribute to the composition of the PC species profile, the precursor-product relationships between PE and newly synthesized PC were determined at the level of the molecular species by using electrospray ionization tandem mass spectrometry and stable isotope labeling. In vivo labeling of yeast cells for 10 min with [methyl-D3]methionine revealed the preferential methylation of di-C16:1 PE over a range of PE species compositions. A similar preferential conversion of di-C16:1 PE to PC was found in vitro upon incubating isolated microsomes with S-adenosyl[methyl-D3]methionine. Yeast opi3 and cho2 deletion strains were used to distinguish between the substrate selectivities of Cho2p and Opi3p, respectively. Both biosynthetic enzymes were found to participate in the speciesselective methylation with Cho2p contributing the most. The combined results indicate that the selective methylation of PE species by the methyltransferases plays an important role in shaping the steady-state profile of PC molecular species in yeast.

Pathways for phosphatidylcholine biosynthesis in bacteria.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions. Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria. Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway. In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs). In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS. Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities. In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities. Genes from M. loti and L. pneumophila encoding a Pmt or a Pcs activity and the genes from P. aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified. Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis. However, important pathogens such as Brucella melitensis, P. aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.

The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase.

Saccharomyces cerevisiae opi3 mutant strains do not have the phospholipid N-methyltransferase that catalyzes the two terminal methylations in the phosphatidylcholine (PC) biosynthetic pathway. This results in a build up of the intermediate phosphatidylmonomethylethanolamine, causing a temperature-sensitive growth phenotype. An Arabidopsis cDNA library was used to isolate three overlapping plasmids that complemented the temperature-sensitive phenotype. Phospholipid analysis showed that the presence of the cloned cDNA caused a 65-fold reduction in the level of phosphatidylmonomethylethanolamine and a significant, though not equivalent, increase in the production of PC. Sequence analysis established that the cDNA was not homologous to OPI3 or to CHO2, the only other yeast phospholipid N-methyltransferase, but was similar to several other classes of methyltransferases. S-adenosyl-Met:phospho-base N-methyltransferase assays revealed that the cDNA catalyzed the three sequential methylations of phospho-ethanolamine to form phospho-choline. Phospho-choline is converted to PC by the CDP-choline pathway, explaining the phenotype conferred upon the yeast mutant strain by the cDNA. In accordance with this the gene has been named AtNMT1. The identification of this enzyme and the failure to isolate a plant phospholipid N-methyltransferase suggests that there are fundamental differences between the pathways utilized by yeast and by some plants for synthesis of PC.

Inactivation of the gene for phospholipid N-methyltransferase in Sinorhizobium meliloti: phosphatidylcholine is required for normal growth.

In phosphatidylcholine (PC)-containing prokaryotes, only the methylation pathway of PC biosynthesis was thought to occur. However, a second choline-dependent pathway for PC formation, the PC synthase (Pcs) pathway, exists in Sinorhizobium (Rhizobium) meliloti in which choline is condensed with CDP-diacylglyceride. Here, we characterize the methylation pathway of PC biosynthesis in S. meliloti. A mutant deficient in phospholipid N-methyltransferase (Pmt) was complemented with a S. meliloti gene bank and the complementing DNA was sequenced. A gene coding for a S-adenosylmethionine-dependent N-methyltransferase was identified as the sinorhizobial Pmt, which showed little similarity to the corresponding enzyme from Rhodobacter sphaeroides. Upon expression of the sinorhizobial Pmt, besides phosphatidylcholine, the methylated intermediates of the methylation pathway, monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine, are also formed. When Pmt-deficient mutants of S. meliloti are grown on minimal medium, they cannot form PC, and they grow significantly more slowly than the wild type. Growth of the Pmt-deficient mutant in the presence of choline allows for PC formation via the Pcs pathway and restores wild-type-like growth. Double knock-out mutants, deficient in Pmt and in Pcs, are unable to form PC and show reduced growth even in the presence of choline. These results suggest that PC is required for normal growth of S. meliloti.

Plant-exuded Choline Is Used for Rhizobial Membrane Lipid Biosynthesis by Phosphatidylcholine Synthase

Phosphatidylcholine is a major lipid of eukaryotic membranes, but found in only few prokaryotes. Enzymatic methylation of phosphatidylethanolamine by phospholipid N-methyltransferase was thought to be the only biosynthetic pathway to yield phosphatidylcholine in bacteria. However, mutants of the microsymbiotic soil bacterium Sinorhizobium (Rhizobium) meliloti, defective in phospholipid N-methyltransferase, form phosphatidylcholine in wild type amounts when choline is provided in the growth medium. Here we describe a second bacterial pathway for phosphatidylcholine biosynthesis involving the novel enzymatic activity, phosphatidylcholine synthase, that forms phosphatidylcholine directly from choline and CDP-diacylglycerol in cell-free extracts of S. meliloti. We further demonstrate that roots of host plants of S. meliloti exude choline and that the amounts of exuded choline are sufficient to allow for maximal phosphatidylcholine biosynthesis in S. meliloti via the novel pathway.

The Schizosaccharomyces pombe cho1+ gene encodes a phospholipid methyltransferase.

The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Delta) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.

Characterization of phospholipid methylation in rat brain myelin

Highly purified rat brain myelin was solubilized in Triton X-100 and myelin phospholipid N-methyltransferase was characterized. The enzyme activities were separated by isoelectric focusing and ion-exchange chromatography. The phospholipid methyl-transferase has shown at least four peaks of activity with pIapp. values of 4.5, 5.2, 6.2 and 8.4. After affinity purification each of these activities revealed a close set of bands of approx. 65 kDa on SDS/PAGE. These data together with those from preparative SDS/PAGE separations suggested that rat brain myelin contains three acidic and at least one basic phospholipid-methylating isoenzymes and that the major isoenzyme in each case is approx. 65 kDa in size. While the predominant product of the reaction catalysed by all detected isoforms was monomethylated phosphatidylethanolamine, the least acidic isoform (pIapp. 6.2) also formed about 20% phosphatidylcholine, suggesting that these isoenzymes may play different roles in vivo.

S-Adenosyl-L-Methionine–Mediated Enzymatic Methylations in the Rat Retinal Membranes

Enzymatic step-wise methylation of membrane phosphatidylethanolamine (PE) to phosphatidyl-N-methylethanolamine (PME) and then phosphatidyl-choline (PC) has been known to alter membrane properties and responsiveness of cells for activation of receptors by chemical transmitters. Conversion of PE to PME and PME to PC in the presence of S-adenosyl-L-methionine (SAM) are catalyzed by two phospholipid N-methyltransferases, PMT I and PMT II, of which PMT I is the rate limiting enzyme. Retina is a good neuronal model for chemical transmission. However, retina was not studied for PMT activity. Therefore, we studied the rat retina for PMT I activity. Methylation of PE in the rat retinal sonicates was assayed using 3H-SAM (2 microM) at 37 degrees C in Tris-glycylglycine buffer (50 mM, pH 8.0) and methylated phospholipids were extracted with chloroform/methanol/HCl (2/1/0.02, v/v) and separated by thin layer chromatography on Silica Gel G plates. Chromatograms were developed in a solvent system of propionic acid/n-propyl alcohol/chloroform/water (2/2/1/1, v/v). This study gave the following results: (a) the total methylated phospholipids were (M +/- SE, N = 5) 19.90 +/- 4.03 fmol/mg protein/min; (b) the major methylated phospholipid was PME (4.21 +/- 0.68 fmol/mg protein/min; (c) the fatty acid methylesters formed by fatty acid carboxymethylase (FACM) which accumulated in the solvent front amounted to 18.82 +/- 2.84 fmol/mg protein/min. Both PMT I and FACM were inhibited by S-adenosyl-L-homocysteine (I50, 1.2-5 microM). These observations indicate that rat retina contains both PMTs and FACM.

The purification and molecular weight determination of rat liver microsomal phospholipid N-methyltransferase

Conference Article| November 01 1993 The purification and molecular weight determination of rat liver microsomal phospholipid N-methyltransferase William A. Gibbons; William A. Gibbons 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Luma Auchi; Luma Auchi 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Kevin McBride; Kevin McBride 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Alfred N. Fonteh; Alfred N. Fonteh 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Banafshe Larijani; Banafshe Larijani 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Vadim Tsvetnitsky; Vadim Tsvetnitsky 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Margaret M. Anderson; Margaret M. Anderson 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Francis A. Yeboah Francis A. Yeboah 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (4): 449S. https://doi.org/10.1042/bst021449s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation William A. Gibbons, Luma Auchi, Kevin McBride, Alfred N. Fonteh, Banafshe Larijani, Vadim Tsvetnitsky, Margaret M. Anderson, Francis A. Yeboah; The purification and molecular weight determination of rat liver microsomal phospholipid N-methyltransferase. Biochem Soc Trans 1 November 1993; 21 (4): 449S. doi: https://doi.org/10.1042/bst021449s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

Analysis of lipid composition of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pig skeletal muscle.

In search of a general membrane defect hypothesis for malignant hyperthermia syndrome, we analysed the lipid profiles of heavy sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia longissimus dorsi pig muscle. Malignant hyperthermia susceptibility was assessed by halothane challenge of pigs. Sarcoplasmic reticulum membranes from malignant hyperthermia susceptible pigs differed significantly from control ones in the cholesterol content and phosphatidylethanolamine/phosphatidylcholine ratio; both were higher in former membranes. These latter lipid modifications were in agreement with the significant increase of their bulk lipid viscosity, as evidenced by an increase of diphenyl hexatriene fluorescence anisotropy. The increased level of phosphatidylethanolamine associated with the decreased content of phosphatidylcholine in malignant hyperthermic membranes was shown to be a potential consequence of depressed activities of both phospholipid N-methyltransferase I and II activities. Finally, the distribution of fatty acids in these particular phospholipids showed no change in phosphatidylcholine molecules, whereas the percentage of arachidonate and stearate in the phosphatidylethanolamine species were respectively higher and lower in malignant hyperthermic membranes. These differences in major phospholipids content and the enrichment of a metabolically important fatty acyl chains in malignant hyperthermia sarcoplasmic reticulum membranes strongly suggest that the lipid metabolism may contribute to the molecular mechanism of malignant hyperthermia syndrome.

Regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae by inositol and choline: Kinetics of repression and derepression

The biosynthesis of phosphatidylserine (PS) and its conversion to phosphatidylcholine (PC) are regulated coordinately by inositol and choline in Saccharomyces cerevisiae ( G. M. Carman and S. A. Henry, 1989, Annu. Rev. Biochem. 58, 635–669). In this study, PS decarboxylase activity is shown to be partially repressed when inositol is added to the medium of cells in the log phase of growth, and the extent of repression is augmented by the inclusion of choline, but not ethanolamine. The kinetics of repression and derepression of PS decarboxylase, PS synthase, and phospholipid N-methyltransferase (PNMT) activities, as regulatory responses to the availability of exogenous inositol and choline, have been characterized. When inositol was added to the medium of cell cultures growing exponentially, the three biosynthetic enzyme activities reached an intermediate level of repression (50–85% of control) within 60 min. After the addition of the combination of inositol and choline, PS decarboxylase, PS synthase, and PNMT activities decreased to the intermediate levels of repression in 60 min and were subsequently reduced to 15–40% of control values during a later stage of regulation (2–3 h). In a derepression study, the three enzyme activities remained relatively stable for approximately 60 min following the removal of choline and/or inositol from the growth medium, but the specific activities of PS decarboxylase, PS synthase, and PNMT increased to maximally derepressed levels within 2–3 h. The induction of the three biosynthetic activities was blocked by cycloheximide, but not by chloramphenicol. In summary, the level of PS decarboxylase activity in S. cerevisiae is partially and reversibly suppressed by inositol and further diminished by the combination of inositol and choline. The biphasic kinetics of repression by inositol and choline suggest that the effect of choline is dependent on earlier events mediated by inositol and possibly involves a separate regulatory factor(s).

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

Placental toxicology: tobacco smoke, abused drugs, multiple chemical interactions, and placental function

There are increasing numbers of reports on the tobacco smoking and ingestion of abused drugs (e.g. morphine, cocaine) by pregnant women and the effects of the substances on the developing fetus and newborn infant. The passage of drugs and chemicals from the mother to the fetus is influenced by the placental transport and metabolism of the substances. Further, these drugs and chemicals affect the nutrient transport systems in the placenta. The three major drugs of abuse-nicotine, morphine and cocaine-depress both active amino-acid uptake by human placental villi and transplacental amino-acid transport by reason of the drugs' influence on placental cholinergic and opiate systems. Part of this depression (10-16%) is not reversible. Nicotine blocks the cholinergic receptor and thus blocks acetylcholine (ACh)-facilitated amino-acid transport. Morphine stimulates opiate kappa receptors and depresses ACh release. Cocaine blocks Ca2+ influx and thus blocks ACh release. ACh causes dilation of blood vessels and maintains placental blood flow by the activation of endothelial muscarinic receptors. By interfering with ACh release and placental blood flow, the three drugs of abuse may depress the diffusion of amino acids and other nutrients from the trophoblast into the placental circulation. Three regulatory systems are delineated for amino-acid uptake by the placenta: placental ACh, phospholipid N-methyltransferase, and the gammaglutamyl cycle. These systems operate in concert with one another and are dependent on cellular formation of adenosine 5'-triphosphate (ATP). Placental hypoxia induced by carbon monoxide and other tobacco gases depresses the energy-dependent processes and thus the ATP levels of placental cells. Maternal tobacco smoking and drug abuse cause placental insufficiencies for amino-acid transport, which may partially explain the fetal intrauterine growth retardation caused by these substances. Part of the amino-acid deficits may be compensated for by the induction of new amino-acid transport systems. Specific receptors or drug-binding proteins for the three drugs of abuse are present in the placenta. A DNA adduct selective for maternal smoking has been demonstrated in the placenta. DNA adducts selective for cocaine, morphine and other environmental chemicals have yet to be demonstrated ins the placenta.

Cyclic nucleotide phosphodiesterases and methyltransferases in purified lymphocytes, monocytes and polymorphonuclear leucocytes from healthy donors and asthmatic patients

Cyclic nucleotide phosphodiesterase and phospholipid N-methyl-transferase activities were simultaneously measured in purified polymorphonuclear cell-, mononuclear cell-, lymphocyte- and monocyte-homogenates from control subjects, from patients with atopic asthma and from patients with non-atopic asthma. Whereas cyclic AMP and cyclic GMP phosphodiesterase activities were found to be about 10-fold lower in polymorphonuclear than in mononuclear cells, phospholipid N-methyltransferase proved to be rather similar in each cell type from control donors. Cyclic AMP phosphodiesterase and phospholipid N-methyltransferase were significantly decreased in polymorphonuclear cells and monocytes from asthmatic patients compared with the control group while cyclic GMP phosphodiesterase was significantly impaired only in the monocyte subpopulation.