Inhibition Of Phospholipid Synthesis Research Articles

NMDA Receptor Overactivation Inhibits Phospholipid Synthesis by Decreasing Choline–Ethanolamine Phosphotransferase Activity

Overactivation of NMDA receptors is believed to induce neuronal death by increasing phospholipid hydrolysis and subsequent degradation. We showed previously that NMDA releases choline and inhibits incorporation of [3H]choline into phosphatidylcholine before excitotoxic neuronal death. On the basis of these results, we hypothesized that excitotoxicity results from inhibition of synthesis rather than from increased degradation of phospholipids. We now investigated the effect of NMDA receptor overactivation on synthesis and degradation of major membrane phospholipids in the early stages of the excitotoxic process. Exposure of cortical neurons to neurotoxic concentrations of NMDA increased extracellular choline and activated hydrolysis of phosphatidylcholine and phosphatidylinositol by phospholipase A2 but did not induce significant degradation of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, or phosphatidylserine. In contrast, NMDA strongly reduced the incorporation of [3H]choline and [3H]ethanolamine into their respective phospholipids. Metabolic labeling experiments in whole cells showed that NMDA receptor overactivation does not modify the activity of phosphocholine or phosphoethanolamine cytidylyltransferases but strongly inhibits choline-ethanolamine phosphotransferase activity. This effect was observed well before any significant membrane damage and cell death. Moreover, cholinephosphotransferase activity was lower in microsomes from NMDA-treated cells. These results show that membrane damage by NMDA is preceded by inhibition of phospholipid synthesis and not by phospholipid degradation in the early stages of the excitotoxic process, and that NMDA receptor overactivation decreases phosphatidylcholine and phosphatidylethanolamine synthesis by inhibiting choline-ethanolaminophosphotransferase activity.

Alterations in glial cell metabolism during recovery from chronic osmotic stress.

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of 13C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of 13C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of 13C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that 'readaptation' of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.

Role of precursor translocation in coordination of murein and phospholipid synthesis in Escherichia coli.

Inhibition of phospholipid synthesis in Escherichia coli by either cerulenin treatment or glycerol starvation of a glycerol-auxotrophic mutant resulted in a concomitant block of murein synthesis. The intracellular pool of cytoplasmic and lipid-linked murein precursors was not affected by an inhibition of phospholipid synthesis, nor was the activity of the penicillin-binding proteins. In addition, a decrease in the activity of the two lipoprotein murein hydrolases, the lytic transglycosylases A and B, could not be demonstrated. The indirect inhibition of murein synthesis by cerulenin resulted in a 68% decrease of trimeric muropeptide structures, proposed to represent the attachment points of newly added murein. Importantly, inhibition of phospholipid synthesis also inhibited O-antigen synthesis with a sensitivity and kinetics similar to those of murein synthesis. It is concluded that the step common for murein and O-antigen synthesis, the translocation of the respective bactoprenolphosphate-linked precursor molecules, is affected by an inhibition of phospholipid synthesis. Consistent with this assumption, it was shown that murein synthesis no longer depends on ongoing phospholipid synthesis in ether-permeabilized cells. We propose that the assembly of a murein-synthesizing machinery, a multienzyme complex consisting of murein hydrolases and synthases, at specific sites of the membrane, where integral membrane proteins such as RodA and FtsW facilitate the translocation of the lipid-linked murein precursors to the periplasm, depends on ongoing phospholipid synthesis. This would explain the well-known phenomenon that both murein synthesis and antibiotic-induced autolysis depend on phospholipid synthesis and thereby indirectly on the stringent control.

Dependence of peptidoglycan metabolism on phospholipid synthesis during growth of Escherichia coli.

The role of phospholipid synthesis in peptidoglycan metabolism during growth of Escherichia coli was determined. The inhibition of phospholipid synthesis, achieved by inhibiting fatty acid synthesis with cerulenin or by glycerol deprivation of gpsA mutant strains, resulted in the concomitant inhibition of peptidoglycan synthesis. These effects on peptidoglycan synthesis were relatively specific in that the treatments did not cause a general inhibition of macromolecular synthesis. Furthermore, the inhibition of phospholipid synthesis also resulted in the rapid development of penicillin tolerance. It was unlikely that penicillin tolerance in these cases were simply due to the inhibition of growth caused by cerulenin treatment or glycerol deprivation because treatments with more effective growth inhibitors, e.g. chloramphenicol or norfloxacin, did not confer penicillin tolerance. Penicillin tolerance was shown to be a direct consequence of the inhibition of phospholipid synthesis and not due to the possible accumulation of guanosine-3',5'-bispyrophosphate (ppGpp), the starvation stress signal molecule known to be responsible for the development of penicillin tolerance in amino-acid-deprived bacteria. Therefore, peptidoglycan metabolism is coupled to phospholipid synthesis during growth of E. coli, and this may represent an important means to ensure the coordination of cell envelope synthesis in growing bacteria.

Lysophosphatidylcholine Attenuates the Cytotoxic Effects of the Antineoplastic Phospholipid 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine

A colony-stimulating factor 1-dependent cell line was used to determine the relationship between the inhibition of phospholipid synthesis and the cytotoxic activity of the antineoplastic ether lipid, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). ET-18-OCH3 inhibited choline incorporation into phosphatidylcholine as well as total phospholipid synthesis. Exposure to ET-18-OCH3 at the G1/S boundary led to the accumulation of cells in G2, whereas the addition of ET-18-OCH3 in the G1 phase of the cell cycle prevented entry into the S phase. In both cases, ET-18-OCH3 treatment triggered DNA fragmentation and morphological changes associated with apoptosis within 10 h. The addition of lysophosphatidylcholine provided an exogenous source of cellular phospholipid and prevented ET-18-OCH3-dependent accumulation of cells in G2 and apoptosis. However, lysophosphatidylcholine did not overcome the ET-18-OCH3-dependent G1 block, although the growth-arrested cells remained viable. These data indicate that restoring phosphatidylcholine synthesis by supplementation with lysophosphatidylcholine overrides the cytotoxic but not the cytostatic activity of ET-18-OCH3.

Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action.

The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results. We report construction of an E. coli strain that allows valid [14C]acetate labeling of fatty acids under these conditions. In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis. By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth. Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures. Analysis of extracts of [35S]methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid. Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid. The introduction of multicopy plasmids that greatly overproduced either E. coli thioesterase I or E. coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect. Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species. The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s).

THE EFFECT OF LECTINS ON GERMINATING POLLEN OF LILIUM LONGIFLORUM II. EFFECT OF CONCANAVALIN A ON PHOSPHOLIPID TURNOVER AND ON BIOSYNTHESIS OF PECTIC POLYSACCHARIDES

SUMMARY The present study describes the effect of Concanavalin A (Con A, 200 μg/ml) on phospholipid turnover and on the biosynthesis of pectic polysaccharides in germinating pollen of Lilium longiflorum. Both phenomena are directly involved in pollen tube growth. The turnover rate of phospholipids was reduced with about 50% by the presence of Con A during the first 4 hours of germination. When Con A was added after 4 hours of incubation in the absence of lectin, a sudden shift of tritium label was observed from phospholipids to 1,2-diacylglycerol. This shift is probably due to inhibition of phospholipid synthesis during the turnover process, while degradation continues. The biosynthesis of pectic polysacharides was also inhibited with about 50% upon addition of Con A either at the onset of germination or to actively growing pollen tubes which were preincubated for 4 hours in the absence of lectin.

Duplication of Escherichia coli during inhibition of net phospholipid synthesis.

In Escherichia coli BB26-36, the inhibition of net phospholipid synthesis during glycerol starvation affected cell duplication in a manner that was similar in some respects to that observed during the inhibition of protein synthesis. Ongoing rounds of chromosome replication continued, and cells in the D period divided. The initiation of new rounds of chromosome replication and division of cells in the C period were inhibited. Unlike the inhibition of protein synthesis, however, the accumulation of initiation potential in dnaA and dnaC mutants at the nonpermissive temperature was not affected by the inhibition of phospholipid synthesis. Furthermore, proteins synthesized during the inhibition of phospholipid synthesis can be utilized later for division. The results are consistent with a dual requirement for protein and phospholipid synthesis for both the inauguration of new rounds of chromosome replication and the initiation of septum formation. Once initiated, both processes progress to completion independent of continuous phospholipid and protein synthesis.

Methylprednisolone increases the toxicity of oxygen in adult mice. Mechanical and biochemical effects on the surfactant system.

Because previous studies have suggested that some of the toxic effects of oxygen may result from impaired surfactant phospholipid metabolism, we examined the effects of methylprednisolone on the surfactant system in adult mice exposed to greater than 98% O2. Mice pretreated for 7 days with methylprednisolone, 10 mg/kg/day, had a significantly greater mortality in hyperoxia than saline-pretreated, oxygen-exposed control mice. Their bronchoalveolar lavage fluid contained significantly less desaturated phospholipid, and static pressure-volume curves with air and saline showed decreased lung compliance caused by stiffness of the surface layer. The synthesis of desaturated phospholipids by lung slices in vitro was markedly impaired by the first day of hyperoxia, although in the absence of hyperoxia, it was mildly increased. The increased inhibition of phospholipid synthesis in steroid-treated mice was present even when mice were first given steroids on the day of entry into hyperoxia. These results suggest that methylprednisolone has an unfavorable effect on the surfactant system in oxygen toxicity by accentuating the inhibition of phospholipid synthesis. The possibility that these results could have been due to infection promoted by steroid treatment was considered. No cultural, histologic, or serologic evidence in support of this possibility was found, although infection cannot be conclusively excluded.

Regulation of phospholipid synthesis in Escherichia coli. Composition of the acyl-acyl carrier protein pool in vivo.

The regulation of membrane lipid biogenesis was investigated by measuring the levels of the acyl-acyl carrier protein (acyl-ACP) intermediates in the biosynthetic pathway. In particular, the role of the sn-glycerol-3-phosphate acyltransferase was assessed by focusing on the size and composition of the long chain acyl-ACP pool. The ACP( pool was specifically labeled in vivo with beta-[3-3H]alanine and the ACP subspecies analyzed by reversed phase liquid chromatography and conformationally sensitive gel electrophoresis. The acyl-ACP pool was found to be a small fraction of the total ACP in normally growing cells and was particularly devoid of chain lengths that could serve as acyltransferase substrates. Inhibition of phospholipid synthesis at the acyltransferase step resulted in a rapid increase in the content of acyl-ACP, and analysis showed the presence of chain lengths that are acyltransferase substrates. Acyl-CoAs were not detected during interruption of acyl transfer activity. These results show that 1) acyl-ACPs are the acyl donors for phospholipid synthesis in vivo, 2) the acyltransferase does not play a role in the regulation of the lipid biosynthetic rate or the composition of phospholipid acyl moieties, 3) the primary regulatory site in phospholipid biosynthesis is at an early step in fatty acid biosynthesis, 4) feedback regulation by long chain acyl-ACP's is not a controlling mechanism for fatty acid synthesis under normal physiological circumstances, and 5) enzymes that utilize acyl-ACPs are involved in kinetic competition for the scarce acyl-ACP substrates.

Adenylate kinase of Escherichia coli: evidence for a functional interaction in phospholipid synthesis.

Previous genetic and biochemical experiments have suggested that the adenylate kinase of Escherichia coli may be directly involved in phospholipid synthesis through formation of a complex with sn-glycerol-3-phosphate acyltransferase, the membrane-bound enzyme that catalyzes the first step in phospholipid synthesis. In this paper we report direct experiments to test this hypothesis. A mutation within the adenylate kinase structural gene is described that results in a temperature-sensitive phospholipid synthesis (assayed in vivo) and a temperature-sensitive acyltransferase. The adenylate kinase activity of this strain is only minimally altered either in vitro or [as assayed by adenosine 5'-triphosphate (ATP) levels] in vivo. This result demonstrates that the inhibition of phospholipid synthesis is not the result of reduced ATP levels. We report the purification of E. coli adenylate kinase to homogeneity; and find that the addition of homogeneous wild-type adenylate kinase to membranes containing a mutationally altered temperature-sensitive acyltransferase results in thermal stabilization of the acyltransferase activity. Ovalbumin has no such protective effect. Purified E. coli inner membranes contain several proteins that are precipitated by addition of anti adenylate kinase antibody to detergent-solubilized membranes.

Recovery from damage induced by acridine plus near-ultraviolet light in Escherichia coli.

Abstract— Escherichia coli cells treated with sublethal doses of acridine plus near‐UV light exhibit an effective split‐dose recovery response that requires an incubation period of about 30–45 min. Studies of the metabolic requirements for split‐dose recovery revealed the following: (a) DNA synthesis is not required for split‐dose recovery; (b) inhibition of electron transport or protein synthesis reduces the efficiency of split‐dose recovery by about one‐half; (c) inhibition of phospholipid synthesis or cell wall synthesis completely eliminates the split‐dose recovery response. These results suggest an involvement of membrane repair mechanisms in response to damage by acridine plus near‐UV light. Additional evidence for such a process was provided by more direct assays for membrane recovery. It was found that cells treated with sublethal doses of acridine plus near‐UV light are sensitive to low concentrations of detergents, and lose that sensitivity upon incubation. Likewise, treated cells are susceptible to lethal osmotic shock, but can recover from this susceptibility if incubated after treatment but prior to exposure to low osmotic conditions. Based on accumulating evidence, we propose that E. coli cells are capable of repairing membrane damage resulting from exposure to acridine plus near‐UV light.

Lipid‐Synthesis‐Dependent Biosynthesis (or Assembly) of Major Outer‐Membrane Proteins of Escherichia coil

Upon inhibition of fatty acid synthesis in the presence of cerulenin, the uptake of 5' AMP and of other nutrients using similar pore systems can be inhibited as much as 70%. The same effect was observed upon inhibition of phospholipid synthesis after glycerol deprivation in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase. Resumption of both fatty acid synthesis and phospholipid synthesis restores a normal uptake of 5' AMP. The protein composition of the outer membranes, analyzed after pulse radiolabelling by [35S]methionine, was mainly altered in OmpF and OmpC proteins. These proteins are the main porins used by most nutrients like 5' AMP. Whereas the synthesis or the assembly of OmpF protein seems to be more inhibited that that of OmpC protein after inhibition of fatty acid synthesis, the reverse case was observed after inhibition of phospholipid synthesis. No protein produced during inhibition of fatty-acid or phospholipid synthesis is reincorporated into the outer membrane after resumption of these syntheses. These results are discussed with regard to the biosynthesis and assembly of these proteins.

Relation of somal lipid synthesis to the fast axonal transport of protein and lipid

The role of somal lipid synthesis in the fast axonal transport of protein and lipid was examined in vitro utilizing spinal/sciatic nerve preparations of bullfrog. Inhibition of phospholipid synthesis in dorsal root ganglia by the amphiphilic cation, fenfluramine (0.1–2.0 mM) was monitored as decreased incorporation of [ 3H]choline into phosphatidyl choline. This inhibition was directly proportional to a decrease in the amount of [ 3H]protein undergoing fast axonal transport, the two variables being related by a slope close to unity. [ 3H]Choline-labeled lipid undergoing fast transport in the axon was unaffected by inhibition of somal phospholipid synthesis. Levels of fenfluramine up to 1.0 mM had no effect on uptake or incorporation of [ 3H]leucine. Selective exposure of desheathed nerve trunks to 1.0 mM fenfluramine had no effect on [ 3H]protein translocation, indicating that local phospholipid synthesis is not required to maintain ongoing transport in the axon. Inhibition of cholesterol synthesis in the ganglia with the analog 20,25-diazacholesterol also resulted in depression of [ 3H]-protein transport. Since synthesis of both phospholipid and cholesterol are required at the level of the ganglion, it is suggested that the initiation of fast axonal transport of protein is dependent on the assembly of lipoprotein structures in the soma.

Binding of Salmonella minnesota R-form glycolipid mR595 to rat fibroblasts and its effect on cell metabolism and cell behaviour.

Trypsinized normal rat embryo fibroblasts and untrypsinized and trypsinized transformed rat fibroblasts have two orders of binding sites for bacterial glycolipid mR595. The high order sites fix 1--3 micrograms glycolipid mR595/10(5) cells and those of the low order fix about 6 microgram glycolipids mR595/10(6) cells. Ca++ is required for the low order glycolipid mR595 binding to be trypsinized but not to the untrypsinized transformed rat fibroblasts. The low order binding is temperature dependent with the transition temperature lying between 25 and 37 degrees C. Exogenously added ganglioside and glycoproteins contained in the fetal calf serum do not inhibit fixation of glycolipid mR595. Only beta-lipoprotein at high concentrations is slightly inhibitory. Glycolipid mR595 fixation to transformed fibroblast does not alter their morphology and appears to slightly improve cell attachment to substratum. Glycolipid mR595 fixation results in a lengthening of the S-phase of the cell cycle and a reduction in 2-deoxyglucose uptake. Uptake of inorganic phosphate is not affected. Inhibition of phospholipid synthesis is observed in mR595 fixed fibroblasts whereas synthesis of cell surface glycoproteins and the content of cellular gangliosides is not affected.

Changes in state of the surfactant in experimental pneumonia

The state of the lung surfactant in rabbits at different stages of development of experimental pneumonia (3–60 days) was compared with the dynamics of oxidoreductases in the alveolar epithelium and cells of the inflammatory focus of infiltration. In the initial stage (3–7 days) of activation of cell metabolism there was a brief increase in, the intensity of surfactant lipid synthesis, accompanied by relative inhibition of phospholipid synthesis. Later, development of degenerative changes and sclerosis of the parenchyma was accompanied by inhibition of synthesis of all components of the surfactant. The surface activity of the surfactant became stabilized at a low level.

Guanosine tetra- and pentaphosphate synthesis by bacterial stringent factor and eukaryotic ribosomes

AMINO acid starvation of rel+ bacteria1 results in a rapid pleiotropic response involving the cessation of stable RNA synthesis, a decrease in the rate of nucleoside transport, an altered pattern of mRNA synthesis, inhibition of phospholipid synthesis and increased protein turnover. This stringent response2 is rapidly reversed when the amino acid starvation is relieved and is apparently mediated by two unusual nucleotides, guanosine 5′-diphosphate, 3′-diphosphate (ppGpp) and guanosine 5′-triphosphate, 3′-diphosphate (pppGpp)3. These nucleotides are synthesised on the ribosome in the presence of uncharged tRNA, ATP, GTP and an enzyme known as stringent factor3. Mutants which fail to exhibit the stringent response to amino acid starvation with the synthesis of ppGpp pppGpp are known as relaxed mutants4 and in Escherichia coli are mapped at three loci; relA (ref. 5), relB (ref. 6), and rplK (relC) (refs 7 and 8). The synthesis of ppGpp and pppGpp has also been observed in various prokaryotes including blue-green algae9. Here we demonstrate the ability of bacterial stringent factor to be stimulated by eukaryotic ribosomal preparations to synthesise both ppGpp and pppGpp.

Lipids and function of etiochloroplasts after ultraviolet, blue and red light-illumination.

The morphogenesis of whole plants and the development of cell organelles are highly affected or regulated by light-treatment. Chloroplasts normally develop from proplastids when illuminated. In complete darkness, proplastids develop into etioplasts with an assembly of characteristic prolamellar bodies. Light-treatment induces the disappearance of the prolamellar bodies and the development of photosynthetically active thylakoids. Besides chlorophyll, nearly all lipids are already present in etioplasts. Some authors divide the thylakoid lipids into more functional moieties as chlorophylls, carotenoids or lipophilic plastidquinones, and into more structural lipids as the glycerolipids, but it must be said that the function of glycoand phospho-lipids has not yet been elucidated. Further, it is not clear what role the glycerolipids play during physiological plastid development and how the lipid synthesis is regulated. Therefore we performed experiments with etiolated tissues and isolated etioplasts from Hordeum vuIgure L. (cv. Breuns Villa) tQ obtain information about the participation of glycerolipids in etiochloroplast development following continuous illumination. The etiochloroplasts were isolated from illuminated tissues by a density-gradient procedure, the fractions were checked for contamination with other organelles by marker-enzyme measurements. The plastid fraction showed contamination with other material of less than 1 %. In further experiments we used short-time irradiation with different light-qualities for studying regulation phenomena for lipid metabolism. As reported earlier, the lipid composition of etiolated barley seedlings changed by continuous irradiation with white and red light (Tevini & Herm, 1974). Especially within the first 8 h of illumination, a special metabolism of glycerolipids and their fatty acids could be seen. We used light with the following intensities: white light, 0.8 W/mZ; red light, 0.5W/mZ; blue light, 0.8W/mZ. In these early stages of development white, red and blue light enhanced the phospholipid synthesis, whereas the glycolipid synthesis showed the opposite effect (Figs. la and lb). The same holds true when lipid values are related to a constant plastid number (lo9). We infer that a part of the galactolipids as the main constituents of the prolammellar system and plastoglobuli in etioplasts must be hydrolysed before use for thylakoid assembly. Phospholipids may play the role of fatty acid carriers for newly formed galactolipids. Long-wave U.V. light (interference filter with Amax. = 366nm, 0.5 W/mZ) on the other hand enhances the galactolipid synthesis during the first hours of illumination. This may be a reflection of partly inhibited prolamellar-body breakdown and small chlorophyll synthesis, so that all newly formed galactolipids can be measured. Protochlorophyll-absorption measurements show no 'Shibata-shift' meaning that no phytylation takes place under these low light-intensity conditions. In contrast with all other light-qualities U.V. light causes inhibition of phospholipid synthesis during the first hours of illumination and later on even the breakdown of phospholipids can be seen by the formation of lysoglycerides. Blue light, given at the same intensity as red light, induces higher chlorophyll amounts and an earlier appearance of normal chlorophyll a/chlorophyll b ratios than red light. The photosynthetic activity of greening etioplasts, isolated from redand blue-light-illuminated tissues by repeated density-gradient centrifugation, develops somewhat earlier under blue light. Even on a chlorophyll basis the dichlorophenolindophenoland ferricyanide-reduction rates of blue-light-illuminated chloroplasts are considerably higher than those of red-light-illuminated chloroplasts (Table 1). The analysis of the ultrastructure of etiochloroplasts (M. Tevini & K. Herm, unpublished work) proves the earlier appearance of multilayered grana stacks ,

Regulation of membrane phospholipid synthesis by the relA gene: dependence on ppGpp levels.

A series of experiments using a pair of isogenic rel+ strains of Escherichia coli differing only in the spoT locus has demonstrated a quantitative correlation between the inhibition of phospholipid synthesis and the intracellular level of ppGpp. The conditions examined were (1) amino acid starvation; (2) release from amino acid starvation; and (3) balanced growth. We also have been shown the presence of a third gene (in addition of relA and spoT) concerned with ppGpp metabolism and have been found that the level of ppGpp during amino acid starvation is unaffected by an increase in the dosage of the relA gene.

Regulation of membrane lipid synthesis in Escherichia coli. Accumulation of free fatty acids of abnormal length during inhibition of phospholipid synthesis.

Glycerol starvation of an Escherichia coli glycerol auxotroph results in a specific inhibition of membrane phospholipid synthesis. Mindich ((1972) J. Bacteriol. 110, 96-102) observed only a trace accumulation of free fatty acid following glycerol deprivation. We have repeated these experiments using glycerol auxotrophs which also possess a lesion in beta oxidation. This defect was introduced in order to control fatty acid degradation. In contrast to the previous results, we find free fatty acid does accumulate during glycerol starvation. Similar results were found using beta oxidation-defective (fadE-) derivatives of both gpsA and plsB glycerol auxotrophs. Upon glycerol starvation of a plsB- fadE- strain, phospholipid synthesis is 90 percent inhibited. Following a lag of 20 to 40 min, free fatty acid synthesis begins and proceeds at a rate that steadily increases until the rate of fatty acid synthesis is equal to that found in glycerol-supplemented cultures. The accumulation of free fatty acid is the result of de novo synthesis. The average chain length of the fatty acid in the unesterified fraction is abnormally long. Two 20-carbon fatty acids, cis-13-eicosenoic acid and arachidic acid, are found in this frction. Furthermore, a greatly increased level of stearic acid and a small amount of a C-22 (behenic) acid are found in the free fatty acid fraction. These data indicate that acyl transfer into phospholipid is a major determinant of phospholipid acyl moiety chain length. Other experiments have shown that the free fatty acid fraction in glycerol-starved cells is metabolically active. This fraction turns over despite the defective beta oxidation system. Restoration of glycerol to starved cells allows the incorporation of the unesterified fatty acids into phospholipid.